Neurosteroids represent a class of endogenous compounds that exert rapid, nongenomic effects through neurotransmitter receptor systems such as gamma-aminobutyric acid(A) (GABA(A)). Two neurosteroids, allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) and pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one), possess anxiolytic and sedative properties and show substitution for ethanol, benzodiazepines, and barbiturates in drug discrimination assays. A previous study examining the discriminative stimulus effects of 10 mg/kg pregnanolone in DBA/2J and C57BL/6J mice showed pregnanolone's discriminative stimulus to be mediated primarily through GABA(A) positive modulation. This study examined the discriminative stimulus effects of a lower training dose (5.6 mg/kg) of pregnanolone in DBA/2J and C57BL/5J mice. Twelve male DBA/2J mice and 12 male C57BL/6J mice were trained to discriminate 5.6 mg/kg pregnanolone. GABA(A)-receptor positive modulators, neuroactive steroids, NMDA receptor antagonists, and 5-HT(3) receptor agonists were tested for pregnanolone substitution. In DBA/2J and C57BL/6J mice benzodiazepine, barbiturate, and GABAergic neuroactive steroids all substituted for pregnanolone. In the DBA/2J mice, NMDA receptor antagonists showed generalization to the discriminative stimulus cues of pregnanolone, an effect not seen in the C57BL/6J mice. 5-HT(3) receptor agonists and zolpidem failed to substitute for pregnanolone's discriminative stimulus in either strain. AlloTHDOC and midazolam were more potent in producing pregnanolone-like discriminative stimulus effects in DBA/2J mice. These results provide a comprehensive look at pregnanolone's discriminative stimulus effects in two commonly used strains of mice. The present data suggest GABA(A)-receptor positive modulation as the predominant receptor mechanism mediating the discriminative stimulus effects of pregnanolone. NMDA receptor antagonism was suggested in the DBA/2J mice and may represent a heterogenous cue produced by the lower training dose of pregnanolone.