Two separate forms of glutamic acid decarboxylase, now termed GAD65 and GAD67, are the rate limiting enzymes for synthesis of gamma-aminobutyric acid (GABA). Because of the significance of GABA to neuroendocrine processes, numerous attempts have been made to determine the impact of gonadal steroids on enzyme functioning with inconclusive results. Therefore, we attempted to determine the impact of estradiol on mRNA levels for each form of GAD by quantitative in situ hybridization histochemistry in various brain regions. Ovariectomized rats were treated with estradiol benzoate or oil vehicle on 2 consecutive days and the brains collected on the third day. DNA probes specific for GAD65 and GAD67 were radiolabeled with CTP32 using asymmetric polymerase chain reaction. Results of in situ hybridizations for each probe on alternate sections from the same animals were analyzed for magnocellular preoptic area (McPOA), dorsal medial nucleus of the hypothalamus (DMN), zona incerta (ZI), and midbrain central gray (MCG). In the McPOA, estradiol exerted opposite effects on the frequency distribution of pixels per cell for two GAD mRNA probes, significantly increasing GAD65 (P < .05) and decreasing GAD67 (P < .01; Kolmogorov-Smirnov). In the DMN, estradiol treatment significantly increased GAD67 by 60% (P < .05; two-way ANOVA) but decreased GAD65 mRNA by 73% (P < .01). Note the direction of effects are opposite between McPOA and DMN. In MCG, analysis showed no estradiol effect on GAD mRNA levels/cells, but the proportion of cells expressing detectable levels of GAD65 or GAD67 increased by 33-40% in estradiol-treated rats (chi 2, P < .001).