The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 126.96.36.199) catalyzes the committed step in the cardiolipin biosynthetic pathway. To study the regulation of PGPS in Schizosaccharomyces pombe, we characterized the enzyme biochemically. Maximum activity occurred in the presence of 6 mM Triton X-100 at pH 7.5. The apparent Km values for CDP-diacylglycerol and glycerol 3-phosphate were 130 and 26 microM, respectively. Optimal activity was at 35 degrees C, and enzyme activity was labile above 40 degrees C. Thioreactive agents were inhibitory to PGPS activity. To determine whether S. pombe PGPS is regulated by phospholipid precursors, we examined the time-dependent expression of PGPS upon inositol and choline starvation. Starvation for inositol resulted in a threefold increase in PGPS expression in wild-type cells. In cho1 and cho2 mutants, which are blocked in phosphatidylcholine synthesis, starvation for choline resulted in transient derepression of PGPS expression. In choline auxotrophs starved for inositol, PGPS was derepressed 2.5- to 3-fold in the presence of choline and less or not at all in the absence of choline. This is the first description of PGPS regulation in S. pombe and the first demonstration of inositol-mediated regulation in the inositol-requiring yeast species.