Phospholipids ofMycobacterium phleiATCC 354 are cardiolipin, phosphatidyl ethanolamine, tri- and tetra-acylated dimannophosphoinositide and tetraacylated pentamannophosphoinositide. Contrary to a previous report (Dhariwalet al. 1975, Experientia, 31: 776–778), monomannophosphoinositide was not evident. A linear decrease in total lipid was observed with an increase in the nitrogen content of the medium. An inverse relationship was observed with the carbon supply in the medium. Cells grown on a medium with limited nitrogen contained more saturated fatty acids, whereas cells grown on a medium with limited carbon contained more unsaturated fatty acids. No significant change in phosphatidyl ethanolamine resulted from changing the carbon and nitrogen supply in the medium. Phosphatidylinositol mannosides were the major phospholipids in glycerol-grown cells, but in glucose-grown cells concentrations of phosphatidylinositol mannosides and cardiolipin were the same. Glyceride content of cells increased with an increase in carbon supply and decreased with an increase in nitrogen supply in the medium. Phosphatidylinositol mannosides were the major phospholipid component at 37 °C, whereas cardiolipin was the major phospholipid in cells grown at 27 °C. Among fatty acids, a considerable decrease in palmitic acid with a concomitant increase in palmitoleic and oleic acid was observed with a decrease in growth temperature from 37 to 27 °C. The results show that there is a preferential production of certain lipid constituents under various cultural conditions.