Changes in spontaneous spike activities from murine frontal cortex networks grown on microelectrode arrays were used to determine the dissociation constants of three GABA(A) antagonists: gabazine, bicuculline, and trimethylolpropane phosphate (TMPP). Networks were treated with fixed concentrations of antagonists and titrated with the GABA(A) receptor agonist muscimol. Muscimol decreased spike activity in a concentration-dependent manner with full efficacy (100% spike inhibition). A sigmoidal curve fit provided a 50% inhibitory concentration (IC(50)) of 0.14+/-0.05muM (mean+/-S.D., n=5). Increasing concentrations of the three antagonists shifted the muscimol concentration response curves (CRCs) to the right with the same efficacy. Schild plot analyses with linear regressions resulted in slopes that are statistically not different from unity and provided X-intercepts (dissociation constants) of 0.23, 0.61, and 3.98muM for gabazine, bicuculline, and TMPP, respectively. Corresponding pA2 values (-logK(B)) were 6.64, 6.21, and 5.40. The dissociation constants for gabazine and bicuculline agree well with those obtained with other methods. The TMPP K(B) has not yet been reported in the literature. The data suggest that spontaneously active networks on microelectrode arrays can be used as reliable platforms for rapid quantitative pharmacological investigations.