We have investigated the rapid steroid effects on intracellular calcium ([Ca2+]i levels in a clonal pituitary cell line (GH3). Among the steroids tested only pregnenolone sulfate induced a rapid and transient [Ca2+]i increase within 1 min. The specificity of pregnenolone sulfate-induced [Ca2+]i increase with respect to steroid structure was pronounced. Other steroids (5-40 microM) including pregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, progesterone, estradiol-17 beta, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-dihydrotestosterone, 5 alpha-dihydroprogesterone, and 3 alpha,5 alpha-tetrahydroprogesterone were found to be ineffective. The [Ca2+]i increase with pregnenolone sulfate (30 microM) was completely abolished in a Ca(2+)-free medium or in the presence of La3+ (0.1 mM) and Co2+ (5 mM). The organic Ca2+ channel blockers methoxyverapamil (100 microM) and nicardipine (5 microM) both showed similar inhibitions (> 73%). The interaction between pregnenolone sulfate and voltage-gated Ca2+ channels (VGCC) was shown by coapplication of pregnenolone sulfate (10 microM) with Bay K 8644 (0.1 mM) or KCl (15 mM). Coapplication of pregnenolone sulfate with KCl increased the [Ca2+]i in an additive manner. However, with the specific agonist Bay K 8644(+/-), the pregnenolone sulfate effect was potentiated in a majority of the cells, suggesting cooperative interaction between the two. The results demonstrate that pregnenolone sulfate induces a rapid Ca2+ influx in GH3 cells. The marked nicardipine block also suggests that most of the Ca2+ influx is mediated through L-type VGCC.