Cultured rat cerebellar granule neurons exposed to solutions of reduced osmolarity, responded initially by swelling followed by a regulatory volume decrease (RVD) which is completed within 15 min. Increasing external osmolarity lead to cell shrinking but no evidence of volume regulation was observed within 1 hr. Replacing Na+ by choline did not affect RVD whereas N-methyl-D-glucamine accelerated the volume recovery and K+ suppressed it completely. The blockade of RVD in high extracellular K+ was only observed when chloride and nitrate but not sulfate or gluconate were the accompanying anions. Replacing intracellular Cl-, by long incubations with gluconate, markedly inhibited RVD. Removal of extracellular Ca2+ or addition of dantrolene which blocks Ca2+ released from intracellular stores had no effect on RVD. Increasing extracellular taurine prevented RVD. These results indicate that membrane permeability to K+, Cl-, and taurine is increased by hyposmolarity and suggest the involvement of these molecules in RVD in granule neurons.