Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [(3)H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.