Effects of gangliosides GM3, GD3 and GT1b on protein kinase C (PKC) activity and endogenous protein phosphorylation by PKC were examined in bovine mammary gland. The gangliosides inhibited PKC activity in both cytosolic and total particulate fractions with IC 50 values (concentrations causing 50% inhibition) of 100–115 μM (GM3), 75–80 μM (GD3) and 20–28 μM (GT1b). Several proteins were shown to be substrates for PKC by phosphorylation in the absence or presence of the PKC cofactors 1-oleoyl-2-acetyl-sn-glycerol (OAG, 5 μg/ml), phosphatidylserine (PS, 25 μg/ml) and Ca 2+ (1 μM). GM3 at 100 μM suppressed phosphorylation in cytosol of 91 kDa and 89 kDa proteins. Similar suppression of phosphorylation of these proteins was achieved by the addition of 50 μM GD3 and 10 μM GT1b; thus the order of suppressive capacity for substrate phosphorylation was parallel to their IC 50 values. GT1b (100 μM) inhibited phosphorylation of 56 kDa, 43 kDa and 36 kDa proteins, in addition to that of the 91 kDa and 89 kDa ones. Phosphorylation of 72 kDa and 21 kDa proteins was resistant even at this concentration, suggesting the selective suppression by gangliosides of phosphorylation of individual substrates. In the particulate fraction, phosphorylation of a 91 kDa protein was suppressed by 100 μM GT1b, wheres phosphorylation of the other substrates was unaffected by any ganglioside used. Suppression of phosphorylation of cytosolic 91 kDa and 89 kDa and particulate 91 kDa proteins could be reversed by addition of PS (250 μg/ml), but not by OAG (50 μg/ml) or Ca 2+ (250 μM). These results suggest that gangliosides are involved in the regulation of PKC-dependent phosphorylation by modulating the association of PKC or its substrates with membrane phospholipids.