Phosphatidic acid is the biosynthetic precursor of all glycerolipids. To understand how phosphatidic acid biosynthesis is controlled in Saccharomyces cerevisiae, we studied the regulation of three enzyme activities involved in the synthesis of this glycerolipid precursor, i.e., glycerophosphate acyltransferase (GPAT), dihydroxyacetone phosphate acyltransferase (DHAPAT), and acyl DHAP reductase. GPAT activity was increased 3-fold, while DHAPAT activity was increased up to 9-fold in wild type cells grown in a nonfermentable carbon source compared to that of glucose-grown cells. The ratio of GPAT/DHAPAT activity was 12 in glucose-grown cells but only 4 in cells grown in glycerol/ethanol. In the previously characterized tpa1 mutant, (T. S. Tillman and R. M. Bell. 1986. J. Biol. Chem. 261: 9144-9149), GPAT was decreased 2-fold and DHAPAT 27-fold compared to activities in the wild type. Acyl DHAP reductase activity in both wild type and tpa1 cells grown on a nonfermentable carbon source was increased approximately 2-fold over that of glucose-grown cells. All three enzymatic activities increased as wild type cells grown on glucose entered the stationary phase of growth. Therefore, GPAT, DHAPAT, and acyl DHAP reductase activities appear to be regulated by the respiratory state of the cell. None of the activities was affected to a great extent by inositol, which is a key regulator of many enzymes involved in the synthesis of PtdOH-derived phospholipids in S. cerevisiae, nor by deletion of the mitochondrial genome.(ABSTRACT TRUNCATED AT 250 WORDS)