Although the acylation of 1-alkenylglycerophosphocholine in mammalian heart is well documented, the acylation of 1-alkenylglycerophosphoethanolamine in the heart was not reported. In this study, the presence of acyl CoA: 1-alkenylglycerophosphoethanolamine acyltransferase in the guinea pig heart microsomes was demonstrated. 1-Alkenylglycerophosphoethanolamine acyltransferase displayed a high degree of specificity towards acyl-CoA. The order of reactivity with acyl-CoA was found to be: linoleoyl much greater than arachidonyl greater than palmitoyl greater than stearoyl = oleoyl. 1-Acylglycerophosphoethanolamine acyltransferase in the microsomes also exhibited specificity towards acyl-CoA in the following manner: linoleoyl greater than arachidonyl much greater than palmitoyl greater than oleoyl greater than stearoyl. However, such specificity appeared to be dependent on acyl-CoA concentration. The acyl-CoA specificities of both enzymes did not correlate with the C-2 acyl distribution observed in the corresponding microsomal phospholipids. Our results suggest that in addition to the acyl specificity of the acyltransferases, intracellular concentrations of acyl-CoAs may also have an important role in determining the observed acyl patterns of the phospholipids. Based on the acyl specificities, pH profiles, and their responses to heat inactivation and thiol reagents, we conclude that 1-alkenylglycerophosphoethanolamine acyltransferase and 1-acylglycerophosphoethanolamine acyltransferase in guinea-pig heart microsomes are not the same enzyme.