The intracellular free Ca2+ concentration was measured in freshly dissociated brain cells prepared from neonatal rats using the fluorescent Ca2+ indicator Fura-2/AM. Cytosolic Ca2+ concentration of resting cells was calculated to be 240 +/- 5 nmol/L. Depolarization with high K+ resulted in an over 100% increase in intracellular Ca2+ concentration, and this increase could be prevented or reversed by verapamil or nifedipine known to block voltage-sensitive Ca channels. These results suggest that the adoption of Fura-2/AM method in freshly dissociated rat brain cells is a useful and relatively easily applicable technique for monitoring intracellular Ca2+ changes.