Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one 3 alpha,5 beta-P), were studied for differences in their pharmacological properties using behavioral assays. 3 alpha,5 alpha-P and 3 alpha,5 beta-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED50: 3 alpha, 5 alpha-P = 2.8 mg/kg and 3 alpha,5 beta-P = 3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD50: 3 alpha,5 alpha-P = 18.8 mg/kg and 3 alpha,5 beta-P = 21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3 alpha,5 beta-P was more efficacious than 3 alpha,5 alpha-P, though both compounds had similar potencies. In the Geller-Seifter test, 3 alpha,5 beta-P was more potent and efficacious than 3 alpha,5 alpha-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3 alpha,5 alpha-P and 3 alpha,5 beta-P have similar anticonvulsant activity, but the 5 beta-isomer possesses more potent and efficacious anxiolytic properties than the 5 alpha-isomer.