To investigate if vitamin B6 inhibits prolactin release and to compare this effect to that of bromocriptine, a known suppressor of prolactin release, a study was conducted in male rats. Animals were pretreated with pyridoxine hydrochloride, pyridoxal hydrochloride, saline, or bromocriptine 30 min prior to receiving varying doses of chlorpromazine hydrochloride. Blood samples were obtained 90 min later and analyzed for serum prolactin by a double-antibody radioimmunoassay. Another study involved pyridoxal hydrochloride and saline pretreatments 30 min prior to doses of chlorpromazine hydrochloride. Blood samples collected 60 min later were also analyzed for serum prolactin. Pyridoxine hydrochloride significantly suppressed the chlorpromazine-induced prolactin rise (p less than 0.01). However, the suppression was significantly less than that produced by bromocriptine (p less than 0.01). Pyridoxal hydrochloride, another natural form of vitamin B6, failed to suppress prolactin under the conditions of both studies. This investigation may lend support to the concept that pyridoxine hydrochloride partially inhibits prolactin by a mechanism not involving dopamine.