The present studies examine the effects of testosterone (T) and LHRH, alone or in combination, on the amount of FSH secreted by pituitary cells in culture. FSH was quantified by RIA and radioreceptor assay (RRA). Half of the cultures were exposed to T for 3 days. The remainder served as controls. Each of these two groups was divided in half and exposed to medium only or LHRH (10(-8) M) for 4 h. Medium was collected from all cultures after 3 days +/- T (medium 1) and after 4 h +/- LHRH (medium 2). After medium 2 collection, cell homogenates were prepared. In a second study, T-treated cell cultures also received 0, 0.5, or 5.0 micrograms/dish tunicamycin for the last 16 h of the 3-day incubation. During the 3 days of culture, the T-treated group secreted greater amounts of immunoactive FSH than controls. However, LHRH-induced FSH release measured by RIA was blunted as a result of T exposure compared with untreated controls. T treatment elevated intracellular immuno-FSH stores. Each sample was quantitated for FSH activity by RRA, and the FSH RRA/RIA was calculated. T and/or LHRH treatment, while eliciting FSH hypersecretion, caused a reduction in the RRA/RIA of secreted FSH. Changes in the RRA/RIA are thought to occur as a result of alterations in the glycosylation of FSH. To test this hypothesis, T-treated cells were exposed to tunicamycin, a drug that reduces the rate of glycosylation of secreted proteins. Exposure of cells to this drug prevented the reduction in the RRA/RIA of secreted FSH caused by T and/or LHRH. FSH secreted from control, T-treated, or T-treated plus tunicamycin-exposed cells was examined by isoelectric focusing. T-Treated cells released a greater proportion of FSH forms with lower isoelectric points (indicative of a greater degree of glycosylation) compared with controls. Tunicamycin exposure reversed T's effect upon the isoelectric profile. These studies demonstrate a direct pituitary action of LHRH and T upon the type of FSH released. During times of hormonally induced increases in the rate of FSH secretion, the pituitary releases FSH forms that are more heavily glycosylated, exhibit a lower isoelectric point, and show a reduced RRA/RIA. FSH secreted after T treatment would be expected to have an increased plasma half-life due to the protective effects of the sugar residues. Thus, the existing hormonal milieu exerts a multidimensional effect upon FSH released by pituitary cells in culture that cannot be appreciated by RIA assessment alone.