Summary We have investigated the enzymic properties of the extraplastidial acyl-CoA: sn-1-acyl-glycerol-3-phosphate acyltransferase in order to determine whether the exclusion of C 16 fatty acids from the C2-position of eukaryotic glycerolipids is due to the action of this enzyme. The assay conditions were optimized using a microsomal membrane preparation as the enzyme source. The enzyme displayed maximal acylation rates at pH 10 with 75 µM 1-acyl-glycerol-3phosphate and 150 µM oleoyl-CoA, reaching specific activities of about 850 pkat/mg protein. As judged from the separation of microsomal membranes on different types of sucrose gradients, most of the microsomal 1-acyl-glycerol-3-phosphate acyltransferase activity was located in the endoplasmic reticulum. Substrate specificities and selectivities of the enzyme were determined. Oleoyl-CoA was used at higher rates than palmitoyl-CoA. Moreover, 1-oleoyl-glycerol-3-phosphate was a more efficient substrate than 1-palmitoyl-glycerol-3-phosphate. From mixtures of oleoyl- and palmitoyl-CoA, the enzyme selectively transferred oleic acid to the C2 position of 1-palmitoyl- and 1-oleoyl-glycerol-3-phosphate. 1-Acyl-glycerol-3-phosphate acyltransferase is less susceptible to inhibition by the hypolipidemic drug DH990 than lysophosphatidylcholine acyltransferase and thus providing a possibility to distinguish these two enzymes.