The effect of osmotic changes on aldosterone production,[ Ca²⁺]_i and voltage-gated Ca²⁺ currents, was studied in cultured rat glomerulosa cells. Alteration of osmolarity by sucrose addition in the 250–330 mosm range did not influence aldosterone production per se, but it substantially affected K⁺-stimulated aldosterone production. Hyposmosis markedly increased the hormone response evoked by raising [K⁺] from 3.6 to 5 mm, whereas hyperosmosis had a mild decreasing effect. Cytoplasmic[ Ca²⁺]_i, measured in single glomerulosa cells, did not show detectable change in response to either hyposmotic or hyperosmotic exposure, but the [Ca²⁺]_i signal evoked by elevation of [K⁺] to 5 mm was augmented in hyposmotic solution. The osmosensitivity of the transient (T)-type and long-lasting (L)-type voltage-gated Ca²⁺ currents was studied using the nystatin-perforated voltage-clamp technique. Lowering osmolarity to 250 mosm significantly increased the amplitude of the T-type current, and it had a transient augmenting effect on L-type current amplitude. Hyperosmotic solution (330 mosm) reduced L-type current amplitude but did not evoke significant change in T-type current. These results indicate that the responsiveness of rat glomerulosa cells to physiological elevation of [K⁺] is remarkably influenced by changes in osmolarity by means of modulating the function of voltage-gated Ca²⁺ channels.