The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated. While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M. gallisepticum, M. pulmonis, and M. pneumoniae was disaturated PC, apparently formed by modification of l-saturated-2-unsaturated PC from the growth medium. The modification of the exogenous PC by M. gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol. A low activity of an endogenous phospholipase A was detected in native M. gallisepticum membranes. The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents. The PC modification was affected by the fatty acid composition of the exogenous PC species. Diunsaturated, l-saturated-2-unsaturated, and l-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not. Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent. The possibility that the incorporation of DPPC into M. gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.In the absence of a rigid cell wall (19), the cell membrane of mycoplasmas interacts directly with components of the growth medium. Most mycoplasmas are fastidious organisms requiring serum for growth (19,30). Direct interaction of Mycoplasma or Spiroplasma species with the serum lipoprotein particles or other exogenous lipid donors results in the incorporation of exogenous lipid from the growth medium (6,18,20,25). Since the major phospholipids in serum-containing growth medium are phosphatidylcholine (PC) and sphingomyelin (SPM), these phospholipids are the major phospholipids incorporated (20). Some Mycoplasma species incorporate more PC than SPM (20), whereas others, as well as all the Spiroplasma species tested, incorporate more SPM (6,20). The incorporated lipids are intercalated into the bulk lipid bilayer (27). In M. capricolum the incorporation of exogenous PC was found to alter the de novosynthesized phospholipids as well as the physical properties of the membrane (11), and more recently, the ability of mycoplasmas to incorporate exogenous phospholipids was correlated with their ability to take up cholesterol (8). In M. gallisepticum, cell growth was found to be greatly stimulated by the incorporation of exogenous SPM (22). The incorporation of exogenous phospholipids was first investigated by us in M. gallisepticum (25) in which it was found that the SPM was incorporated unchanged whereas the PC was modified by the cells. The modification involves the replacement of the unsaturated fatty acid that occupies position 2 of the serum PC with a saturated fatty acid giving rise to a disaturated PC.In this report we show that the modification of exogenous PC ...