Basal plasma prolactin concentration is controlled by tonic inhibition. The major prolactin-inhibiting factor (PIF) is believed to be dopamine. Factors other than dopamine have also been suggested as possible physiological PIF. One of the major candidates for the nondopaminergic PIF is considered to be gamma-aminobutyric acid (GABA). We have carefully examined the possible physiological role of GABA by monitoring, at every 2 min, the circulating prolactin concentration after GABA administration, in conscious freely moving rats. GABA (0.1 or 1 g/kg) had no significant direct effect on plasma prolactin in rats in which the dopaminergic receptors were completely blocked by pimozide, nor in hypophysectomized rats in which a pituitary had been grafted under the kidney capsule and was therefore removed from any hypothalamic influence. The effects of bicuculline, a GABA-receptor-blocking agent, was examined in order to find out whether a tonic inhibition is exerted by GABA after elimination of tonic dopaminergic inhibition on prolactin secretion. The pimozide-treated rat in which the dopaminergic tone is completely eliminated did not show any prominent elevation of plasma prolactin concentration after bicuculline (300 µg/kg) administration. However, GABA did have an inhibitory effect in a primary pituitary cell monolayer culture system. Therefore, we conclude that GABA does not play a significant role as a physiological PIF and that the inhibitory effect of GABA in vitro is of a pharmacological nature