There is ample evidence of calcium being an intracellular second messenger during volume regulatory processes in various cells including inner medullary collecting duct (IMCD) cells. Therefore, we measured intracellular calcium concentrations (Cai) under anisotonic conditions in primary cultures of IMCD cells using the Fura-2 technique. Basal steady-state calcium at 600 mosmol/l was found to be 110 +/- 4 nmol/l; n = 119. Exposure to hypotonic medium (300 mosmol/l, reduction of sucrose) resulted, within 1 min, in a strong increase in calcium to 563 +/- 87 nmol/l (n = 7; P < 0.01), followed by a decrease over 4-6 min to twice the initial values. The calcium increase was smaller (260 +/- 14 nmol/l; n = 5; P < 0.05) when the osmotic pressure was decreased by reducing NaCl instead of sucrose. Stepwise reduction of osmolarity to either 500 or 400 mosmol/l increased calcium by a significantly smaller extent, suggesting a threshold for calcium influx between 400 and 300 mosmol/l. In hypotonic calcium-free solutions no significant increase in calcium was observed. Verapamil (40 mumol/l), D-600 (40 mumol/l), diltiazem (40 mumol/l), and nifedipine (40 mumol/l) inhibited the hypotonically induced calcium influx in decreasing order of potency. Lanthanum (La3+) and gadolinium (Gd3+) had no effect. Membrane depolarization by incubation in potassium-rich solution diminished calcium influx. Preincubation with cytochalasin B (50 mumol/l for 30 min) resulted in a lower basal calcium level and attenuated the calcium increase during hypotonic shock. These results demonstrate an increased calcium influx during hypotonic shock in IMCD cells in culture mediated by channels whose nature (stretch activated and/or voltage dependent) remains to be determined. The transient increase in Cai in turn may trigger inorganic and organic osmolyte fluxes observed previously.