In ischemic myocardium the time course of nonesterified fatty acid (NEFA) accumulation was studied in relation to changes in regional metabolism and mechanics. In open-chest dogs a coronary artery was partially occluded for 120 min. In the ischemic myocardium no increase was observed in NEFA content within 10 min, whereas changes were found in regional shortening, high-energy phosphate content, and glucose arteriologcal venous difference. During prolonged ischemia NEFA content increased, the highest values being found in the inner and middle layers after 120 min (112 and 85 nmol X g-1, respectively; control values 30); the value in the outer layers after 60 min was 93 nmol X g-1. After 120 min of ischemia, accumulation of NEFA generally occurred when myocardial blood flow was below 0.3 ml X min-1 X g-1 and ATP content was below 10 mumol X g dry wt-1. Under these circumstances the individual NEFA with the highest relative increase was arachidonic acid. The present findings indicate that the changes in mechanical function and metabolism, as observed in myocardium rendered ischemic for 10 min, are not caused by increased NEFA content and that NEFA accumulation may partly result from hydrolysis of glycerophospholipids.