Heat stable adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PK) inhibitor was extracted and partially purified from human heart tissue. One unit of inhibitor was defined as the amount necessary to produce 50% inhibition of the PK activity of 50 p mole/min. The eluate from Sephadex G-75 showed a specific activity of 41 units/mg, with purification of 390-fold and recovery of 23%. The molecular weight was 29,000 by gel filtration and S20,w was 1.4, similar to that of rabbit skeletal muscle PK inhibitor. At the purification step involving trichloracetic acid precipitation, the specific activity was not significantly different between tissue from the atria and ventricles. However, the particulate fraction of ventricular homogenate yielded a specific activity that was 3 times higher than that of the supernatant. Kinetic analysis showed that the inhibition was noncompetitive with respect to ATP, histone and cAMP. In the presence of inhibitor the binding of cAMP to PK was increased. This is consistent with the concept that the inhibitor directly interacts with the catalytic subunit of PK, and modifies the physiological action of cyclic AMP.