We examined ganglioside modulation of the activity of the millimolar Ca2(+)-sensitive form (mCANP) of calcium-activated neutral proteinase (CANP), which is enriched in myelin, from brain. GM1, GD1a, GT1a, GM2, and GM4 produced a concentration-dependent increase of mCANP activity. GD1a stimulated the greatest increase of enzyme activity (107%), followed by GT1a, whereas GD1b was inhibitory (56%). GM1, GM2, and GM4 stimulated but less so than GD1a and GT1a. Free N-acetylneuraminic acid, asialo-GM1, GM3, and a ganglioside mixture containing GM1, GD3, GD1a, and GD1b had no effect. The ganglioside-mediated modulation was not affected by trifluoperazine and chlorpromazine (phospholipid-binding antagonists). The mCANP Ca2+ requirement was significantly reduced in the presence of stimulatory gangliosides, and this increased sensitivity varied (10-50-fold) with ganglioside structure. Gangliosides may interact with membrane mCANP and modulate its proteolytic action.