Cholinephosphotransferase is an important enzyme in the synthesis of pulmonary surfactant, the major surface-active component of which is dipalmitoylglycerophosphocholine. Cholinephosphotransferase from rabbit lung microsomes was assayed using a variety of exogenous diacylglycerols as substrate. The enzyme exhibited a preference for unsaturated diacylglycerols, which is in keeping with other evidence that the bulk of pulmonary dipalmitoylglycerophosphocholine is not synthesized de novo. However, 1,2-dipalmitoylglycerol was utilized as a substrate. The highest activity of cholinephosphotransferase was achieved with 1,2-diacylwere 12–16 mg/ml. 1,2-Dioleoylglycerol and 1,2-dipalmitoleoylglycerol were only slightly less effective. The order of preference of cholinephosphotransferase towards other unsatirated diacylglycerols was as follows: 1-stearoyl-2-linoleoylglycerol > 1,2-dilinolenoylglycerol > 1,3-dioleoylglycerol > 1,2-dilinoleoylglycerol > 1-stearoyl-2-oleoylglycerol. These differences in specificity do not appear to be due to differences in diacylglycerol solubility or molecular orientation since the order of preference was unaffected by changes in pH or temperature.