The accumulation of [3H]inositol phosphates derived from phosphoinositide hydrolysis stimulated by gamma- and delta-hexachlorocyclohexane isomers was characterized in primary cultures of cerebellar granule cells. The EC50 for gamma- and delta-hexachlorocyclohexane was 106 and 85 μM, respectively. Stimulatory effects of hexachlorocyclohexane isomers were highly dependent on extracellular Ca2+ but they were not inhibited by classical voltage-sensitive Ca2+ and Na+ channel blockers. The Na+/Ca2+ exchanger blocker amiloride caused a significant inhibition of delta-hexachlorocyclohexane effects. A lack of additive effects on phosphoinositide hydrolysis stimulation between hexachlorocyclohexane isomers and depolarization by high K+ was observed. The effects of each hexachlorocyclohexane isomer on glutamate or carbachol-induced inositol phosphate stimulation were also not additive, whereas that of high K+ was less than additive or synergistic when combined with glutamate or carbachol, respectively. When neuronal cells were exposed to the combination of delta-hexachlorocyclohexane and glutamate or carbachol in the presence of the respective receptor antagonists only the delta-hexachlorocyclohexane stimulatory effect was observed. Thus, the inhibition of glutamate- and carbachol-stimulated phosphoinositide hydrolysis by delta-hexachlorocyclohexane seems to imply a receptor-independent mechanism. It is suggested that both gamma- and delta-hexachlorocyclohexane activate phosphoinositide-specific phospholipase C partly through Ca2+-related mechanisms.