The genome of the yeast, Saccharomyces cerevisiae, contains three highly homologous genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preference in vitro and relative contribution to phospholipid catabolism in vivo (Merkel, Fido, Mayr, Pruger, Raab, Zandonella, Kohlwein, Paltauf (1999) J Biol Chem, 274, 28121-28127). It is shown in this study that in vitro pH strongly affects substrate preference of phospholipase B1 (Plb1p) and phospholipase B2 (Plb2p), but not of phospholipase B3 (Plb3p). At the pH optimum of 2.5-3.5 the substrate preference of Plb1p and Plb2p is PtdSer>PtdIns>PtdCho>PtdEtn. At pH 5 and higher the substrate preference changes to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns vs. PtdCho breakdown, as reflected in the ratio GroPIns/GroPCho released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p because Plb2p does not contribute to PtdIns and PtdCho degradation in vivo (Merkel, Fido, Mayr, Pruger, Raab, Zandonella, Kohlwein, Paltauf (1999) J Biol Chem, 274, 28121-28127). Di- and trivalent cations activate phospholipases B at pH 5.5, but inhibit at pH 2.5. Al 3+ at 20 mM concentration increases Plb1p activity in vitro 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo cycoheximide (CHI) strongly inhibits breakdown of PtdIns and to a lesser extent of PtdCho. However, Al 3+-stimulated GroPCho release is almost completely inhibited by CHI. Deletion of PLB3 leads to increased sensitivity towards toxic Al 3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by CHI. Deletion mutants defective in the PLB1 gene are significantly more resistant towards SDS than wild type cells.