The effect of gamma-aminobutyric acid (GABA) on intracellular Ca2+ concentration ([Ca2+]i) in cultured prenatal rat cortical neurons was investigated using fluorescence imaging. GABA or muscimol, but not baclofen, increased [Ca2+]i in a dose-dependent manner. The GABAA receptor antagonists, bicuculline and picrotoxin, inhibited the GABA response. Furosemide, an inhibitor of the Na+/K+/2Cl- cotransporter, inhibited the GABA response in a noncompetitive manner. Ethacrynic acid, an inhibitor of an ATP-dependent Cl- pump, also inhibited the GABA-induced increased in [Ca2+]i. These results suggest a role for Cl- transport processes in the GABA response. The coapplication of GABA and high K+ led to a non-additive increase in the GABA response. The GABA response was also inhibited by nifedipine, a voltage-gated Ca2+ channel blocker, and abolished by the absence of extracellular Ca2+. Results indicate that the GABA response shares a common pathway of Ca2+ movement with the high K(+)-induced response. These observations suggest that the stimulation with GABA results in Ca2+ influx through voltage-gated Ca2+ channels, and that these effects are dependent on Cl- transport systems.