This study compares the effect of the growth phase on the phospholipid composition and the activity of several phospholipid biosynthetic enzymes in a wild-type yeast grown in fermentable (glucose) and non-fermentable (lactate) semi-synthetic and complete synthetic media. Several distinct differences as well as similarities were found. The cellular phosphatidylcholine: phosphatidylethanolamine (PC : PE) ratio was found to vary with the growth phase, with increases in PC levels at the expense of PE during the transition to stationary phase. The variation was most pronounced in semi-synthetic lactate medium, which is routinely used for the isolation of mitochondria, where the PC : PE ratio changed from 0.9 to 2.2 during this transition. Similar growth phase-dependent changes in PC and PE content were observed in isolated organelles such as mitochondria, mitochondriaassociated membranes and microsomes. Phosphatidylinositol (PI) levels were much higher in cells grown on lactate compared to cells grown on glucose (20% vs. 5±10%). Irrespective of the medium, PI levels increased upon entering stationary phase. The activities of the phospholipid biosynthetic enzymes phosphatidylserine synthase and the phospholipid-Nmethyltransferases were found to be maximal at the end of logarithmic growth and to decrease upon entering stationary phase in all media. Cells grown on lactate displayed a signi®cantly higher phospholipid : protein ratio than cells grown on glucose. The results are discussed in terms of regulation of phospholipid biosynthesis and membrane biogenesis in response to growth phase and carbon source.