Rabbit myocardial cytosolic acyl coenzyme A (acyl-CoA) hydrolase activity was purified to near-homogeneity by ammonium sulfate precipitation and ion-exchange, gel filtration, chromatofocusing, and hydroxylapatite chromatographies. Kinetic analysis of the purified protein demonstrated a maximum velocity of 24 µmol/(mg·min) and an apparent Michaelis constant of 50 µM. Cytosolic acyl-CoA hydrolase and lysophospholipase activities cochromatographed in every fraction of every step. The purified protein was a single band (Mr 23000) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. These results suggest that cytosolic lysophospholipase and palmitoyl-CoA hydrolase activities are catalyzed by a single polypeptide with dual activities. Palmitoyl-CoA competitively inhibited lysophospholipase activity(Ki = 4 µM). Low concentrations (20 µM) of lysophosphatidylcholine or l-palmitoylcarnitine increased palmitoyl-CoA hydrolase activity at low palmitoyl-CoA concentrations but had little effect at high concentrations of palmitoyl-CoA. In contrast, high concentrations (100 µM) of lysophosphatidylcholine or l-palmitoylcarnitine inhibited palmitoyl-CoA hydrolase activity. The results suggest that interactions between endogenous cardiac amphiphiles and palmitoyl-CoA hydrolase contribute to the regulation of intracellular long-chain acyl-CoA concentrations and therefore potentially modulate fluxes of fatty acid through several biochemical pathways.