The effect of cytochalasin B on phosphoinositide (PI) hydrolysis was examined in rat hippocampal slices. Pretreatment of the slices with cytochalasin B caused a significant decrease in PI hydrolysis elicited by carbachol, norepinephrine, or by high K+. This effect was cytochalasin B dose- and time-dependent and was not mimicked by cytochalasin D, vinblastine, colchicine, or phloretin. In contrast, in [3H]inositol-prelabeled hippocampal membranes, cytochalasin B did not affect PI hydrolysis elicited by GTP gamma S and GTP gamma S plus carbachol. Similar result was obtained using the membranes prepared from the slices pretreated with cytochalasin B. The inhibitory effect of cytochalasin B on the carbachol-response was observed in SK-N-SH human neuroblastoma cells, but not in cultured rat astrocytes. These results indicate that cytochalasin B inhibits PI hydrolysis in neuron-specific manner and that its action may be an indirect cellular mechanism other than interaction with cytoskeleton elements.