The relative deacylation of the 1-palmitoyl and 1-stearoyl homologues of different molecular species of endogenous phosphatidylethanolamine in rat liver microsomes via phospholipase activity was studied. For this purpose, the various molecular species of microsomal phosphatidylethanolamine were labelled specifically in the 1-position by incubation of rat liver microsomes with [14C]palmitoyl-CoA or [14C]stearoyl-CoA plus 2-acyl-sn-glycero-3-phosphorylethanolamine containing 18:1, 18:2, 20:4, 22:6, etc. followed by resuspension of the microsomal pellet. The loss of radioactivity from the total 1-[14C]palmitoyl and 1-[14C]stearoyl homologues of phosphatidylethanolamine amounted to 31 and 29%, respectively, when these microsomal preparations were incubated for 1 h in 50 mM Tris-HCl buffer (pH 8.5) containing 10 mM Ca2+. Regardless of whether palmitate or stearate resided in the 1-position, the susceptibility of the phosphatidylethanolamines to deacylation was not influenced significantly by the nature of the unsaturated fatty acid in the 2-position, as judged by selectivity indices for the relative disappearance of radioactivity from the individual classes (monoenoic, dienoic, trienoic, tetraenoic, pentaenoic and hexaenoic). A moderate discrimination against 1-stearoyl 2-saturated species was indicated. The findings indicate that fatty acid selectivity in the microsomal deacylation of phosphatidylethanolamine cannot account for the unique fatty acid and molecular species composition of this phospholipid in rat liver.