The lipid content of Mycoplasma gallisepticum depended on the growth phase of the culture, being high in cells harvested at the early logarithmic phase of growth and low in stationary phase cells. The phospholipid fraction is comprised of three major compounds, tentatively identified as sphingomyelin (SPM), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The relative amounts of the three phospholipids depended on the concentration of horse serum in the growth medium. When grown with increasing serum concentrations, the relative content of PG decreased while that of PC and SPM increased. The SPM of M. gallisepticum appears to be incorporated unchanged from the growth medium. The PC of M. gallisepticum is a disaturated PC, differing from the 1-saturated, 2-unsaturated PC found in the growth medium. The disaturated PC is synthesized by the insertion of a saturated fatty acid at position 2 of lyso-phosphatidylcholine (lyso-PC), derived from exogenous PC of the growth medium, by what appears to be a deacyla-tion-acylation enzymatic sequence. PG is the only phospholipid de novo synthesized by the organisms. It has an unusual positional distribution of fatty acids. Fatty acids with lower melting points are located primarily at position 1 and fatty acids with higher melting points at position 2 of the sn-glycerol 3-phosphate. © 1979, American Chemical Society. All rights reserved.