This paper reports the detailed composition of molecular species of the phosphatidylcholines (PCs) in pulmonary surfactant from calves. PC isolated by thin-layer chromatography (TLC) was converted to benzoylated diradyl glyceride derivatives, which were separated by TLC according to linkage group. Quantification of linkage groups by analysis of total fatty acid content demonstrated that surfactant PC contained 97.2% diacyl, 2.4% alkyl-acyl, and 0.4% alkenyl-acyl compounds. The diacyl and alkyl-acyl diglyceride derivatives were separated into individual molecular species by high-performance liquid chromatography. Four major species constituted 87% of the diacyl compounds. Dipalmitoyl phosphatidylcholine (DPPC) was the most abundant constituent, contributing 41% of the total PC. A second disaturated species, palmitoyl-myristoyl phosphatidylcholine (PMPC), also contributed an additional 12% of total PC. At least 65% of PMPC occurred as the 1-palmitoyl-2-myristoyl/isomer, which has a lower melting point than the 1-myristoyl-2-palmitoyl compound. These results show that most of pulmonary surfactant PC is a relatively simple mixture, that numerous minor compounds are present in small but possibly important amounts, and that in surfactant from calves, the widely reported estimate that DPPC constitutes 60% of surfactant PC is too large by 50%.