The Royal Swedish Academy of Sciences said the two researchers had made groundbreaking discoveries on an important family of receptors, known as G-protein-coupled receptors.
About half of all medications act on these receptors, so learning about them will help scientists to come up with better drugs.
The human body has about 1,000 kinds of such receptors, which let it respond to a wide variety of chemical signals, like adrenaline. Some receptors are in the nose, tongue and eyes, and let us sense smells, tastes and vision.
Lefkowitz, 69, is an investigator at the Howard Hughes Medical Institute and professor at Duke University Medical Center in Durham, North Carolina.
Kobilka, 57, is a professor at Stanford University School of Medicine in California.
"I'm feeling very, very excited," Lefkowitz told a news conference in Stockholm by phone.
He said he was fast asleep when the Nobel committee called.
"I did not hear it ... I wear earplugs, so my wife gave me an elbow," he said. "And there it was. ... It was a total shock and surprise."
Lefktowitz said he had no clue that he was being considered for the Nobel Prize, though he added it has always been "a bit of a fantasy" to receive the award.
Kobilka said he found out around 2:30 a.m., after the Nobel committee called his home twice. He said he didn't get to the phone the first time, but that when he picked up the second time, he spoke to five members of the committee.
"They passed the phone around and congratulated me. I guess they do that so you actually believe them. When one person calls you, it can be a joke, but when five people with convincing Swedish accents call you, then it isn't a joke."
The academy said it was long a mystery how cells interact with their environment and adapt to new situations, such as when adrenaline increases blood pressure and makes the heart beat faster.
Scientists suspected that cell surfaces had some type of receptor for hormones.
Using radioactivity, Lefkowitz managed to unveil receptors including the receptor for adrenaline, and started to understand how it works.
Kobilka's work helped researchers realize that there is a whole family of receptors that look alike -- a family that is now called G-protein-coupled receptors.
The award is "fantastic recognition for helping us further understand the intricate details of biochemical systems in our bodies," said Bassam Z. Shakhashiri, president of the American Chemical Society.
"They both have made great contributions to our understanding of health and disease," Shakhashiri said. "This is going to help us a great deal to develop new pharmaceuticals, new medicines for combating disease."
Mark Downs, chief executive of Britain's Society of Biology, said the critical role receptors play is now taking for granted.
"This ground breaking work spanning genetics and biochemistry has laid the basis for much of our understanding of modern pharmacology as well as how cells in different parts of living organisms can react differently to external stimulation, such as light and smell, or the internal systems which control our bodies such as hormones," Downs said in a statement.
The Nobel week started Monday with the medicine prize going to stem cell pioneers John Gurdon of Britain and Japan's Shinya Yamanaka. Frenchman Serge Haroche and American David Wineland won the physics prize Tuesday for work on quantum particles.
The Nobel Prizes were established in the will of 19th century Swedish industrialist Alfred Nobel, the inventor of dynamite. Each award is worth 8 million kronor, or about $1.2 million. The awards are always handed out on Dec. 10, the anniversary of Nobel's death in 1896.