Long-chain acyl-CoA and acylcarnitine hydrolase activities were determined in fresh and perfused rabbit heart and correlated with tissue levels of their respective substrates, long-chain acyl-CoA and acylcarnitine. In fresh heart homogenate acyl-CoA hydrolase activity was 3-fold greater than acylcarnitine hydrolase activity; sonication of homogenate doubled acyl-CoA hydrolase activity but did not significantly change acylcarnitine hydrolase activity. Hearts perfused with 10 mM glucose by the nonrecirculating Langendorff method had depressed levels of acyl-CoA hydrolase activity under both aerobic and ischemic conditions. Extract from buffer-perfused heart showed increased acylcarnitine hydrolase activity and elevated levels of acylcarnitine. Homogenate acyl-CoA hydrolase activity was not sedimented by centrifugal forces up to 50,000 X g; however, less than 25% of homogenate acylcarnitine hydrolase activity remained in the 50,000 X g supernatant. The hypolipidemic drug clofibrate was an effective in vitro inhibitor of acylcarnitine hydrolase activity but not of acyl-CoA hydrolase activity. The fatty acid analog tetradecylglycidic acid inhibited only acyl-CoA hydrolase activity. These results suggest that acyl-CoA hydrolase and acylcarnitine hydrolase activities are differentially affected in the perfused heart by substrate levels and oxygen availability. In addition, the diverse response of these two hydrolase activities to a variety of biochemical parameters implies that the observed hydrolyses of palmitoyl-CoA and palmitoylcarnitine are catalyzed by at least two separate hydrolase enzymes.