Astroglial swelling occurs in acute hyperammonemic states, including acute hepatic encephalopathy. In these conditions, the peripheral-type benzodiazepine receptor (PBR), a receptor associated with neurosteroidogenesis, is up-regulated. This study examined the potential involvement of PBRs and neurosteroids in ammonia-induced astrocyte swelling in culture. At low micromolar concentrations, the PBR antagonist PK 11195, atrial natriuretic peptide, and protoporhyrin IX, which are known to interact with the PBR, attenuated (16-100%) the effects of ammonia, whereas the PBR agonists Ro5-4864, diazepam binding inhibitor (DBI51-70), and octadecaneuropeptide exacerbated (10-15%) the effects of ammonia. At micromolar concentrations, diazepam, which interacts with both the PBR and the central-type benzodiazepine receptor (CBR), increased swelling by 11%, whereas flumazenil, a CBR antagonist, had no effect. However, at 100 nM diazepam and flumazenil abrogated ammonia-induced swelling. The neurosteroids dehydroepiandrosterone sulfate, tetrahydroprogesterone, pregnenolone sulfate, and tetrahydrodeoxycorticosterone (THDOC), products of PBR stimulation, at micromolar concentrations significantly enhanced (70%) ammonia-induced swelling. However, at nanomolar concentrations, these neurosteroids, with exception of THDOC, blocked ammonia-induced swelling. We conclude that neurosteroids and agents that interact with the PBR influence ammonia-induced swelling. These agents may represent novel therapies for acute hyperammonemic syndromes and other conditions associated with brain edema and astrocyte swelling.