1. Cholinephosphosphotransferase catalyzes the conversion of diacylglycerol and CDPcholine into phosphatidylcholine and CMP. Incubation of rat lung microsomes containing phosphatidyl[Me-14C]choline with CMP resulted in an increase in water-soluble radioactivity, suggesting that also in rat lung microsomes the cholinephosphotransferase reaction is reversible. 2. Microsomes containing 14C-labeled disaturated and 3H-labeled monoenoic phosphatidylcholine were prepared by incubation of these organelles with [1-14C]palmitate and [9,10-3H2]oleate in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine, ATP, coenzyme A and MgCl2. Incubation of these microsomes with CMP resulted in an equal formation of 14C- and 3H-labeled diacylglycerols, indicating that disaturated and monoenoic phosphatidylcholines were used without preference by the backward reaction of the cholinephosphotransferase. When in a similar experiment the phosphatidylcholine was labeled with [9,10-3H2]palmitate and [1-14C]linoleate, somewhat more 14C- than 3H-labeled diacylglycerol was formed. 3. The backward reaction was used to generate membrane-bound mixtures of [1-14C]palmitate- and [9,10-3H2]oleate- or of [9,10-3H2]palmitate- and [1-14C]linoleate-labeled diacylglycerols. When the microsomes containing diacylglycerols were incubated with CDPcholine, both 3H- and 14C-labeled diacylglycerols were used for the formation of phosphatidylcholine, indicating that there is no absolute discrimination against disaturated diacylglycerols. This observation is in line with our previous findings and indicates that also the CDPcholine pathway may contribute to dipalmitoylphosphatidylcholine synthesis in lung.