A quantitative gas chromatography-mass spectrometry (GC/MS) method was developed to measure nanomolar quantities of long-chain saturated beta-hydroxy fatty acids (12, 14, 16, and 18 carbons long) produced by isolated ischemic heart. Only beta-hydroxymyristate (25-40 nmol/g dry) was found in fresh heart. Isolated rabbit heart perfused with fatty acid by the nonrecirculating Langendorff technique produced negligible beta-hydroxy fatty acids. Ischemic perfusion with 0.25-0.75 mM palmitate prompted heart beta-hydroxy fatty acid accumulation, beta-hydroxypalmitate greater than beta-hydroxystearate, up to 100 nmol x g dry-1 x 10 min-1. beta-Hydroxy fatty acid production was proportional to coronary effluent lactate-to pyruvate ratio, did not continue beyond 10 min of ischemia, was dependent on exogenous fatty acid, and was inhibited by coperfusion with 10 mM acetate. Reperfusion for 5-10 min dissipated accumulated beta-hydroxypalmitate. Hypoxic perfusion prompted beta-hydroxy fatty acid production comparable to that with severe ischemia. These data show that during oxygen deficiency heart fatty acid beta-oxidation is not only depressed but is also incomplete; beta-hydroxy fatty acyl intermediates accumulate and contribute to the increased intracellular fatty acid content characteristic of the ischemic myocardium.