To analyze the multihormonal control mechanisms of GTH secretion in the eel, primary culture of pituitary cells from control or estradiol-treated female silver eels, a treatment known to stimulatein vivoGTH synthesis, was developed. Dispersed eel pituitary cells obtained by enzymatic (trypsin/DNAse) and mechanical dispersion were cultured in Earles M199, at 18°. Immunoreactive GTH (ir-GTH) cells were characterized by the immunoperoxidase method, using antiserum to carp GTH beta subunit. Ir-GTH cells from control silver eels were small and represented 14% of the dispersed pituitary cells. In contrast, ir-GTH cells from estradiol-treated eels were larger (cell area ×2.5) and represented a higher proportion (21%) of the pituitary cells. Intracellular and medium contents of GTH were measured by radioimmunoassay for the GTH beta subunit.In vivoestradiol-treatment increased more than 100-fold the GTH content of cell cultures. GTH release, studied over 1 to 4 hr, was undetectable in cultures from normal eels. In contrast, GTH release was low (less than 2% of cell content) but measurable in cultures from estradiol-treated eels. Subsequent experiments examined effects of various secretagogues on GTH release from primary cultures of pituitary cells from estradiol-pretreated eels. GTH release was significantly increased (×1.5 to ×3 basal release) by 10−6MGnRH-A as well as by both native GnRHs in the eel (mammalian GnRH, mGnRH, and chicken GnRH-II, cGnRH-II), at the same concentration. Lower GnRH concentrations had no significant effect, indicating a low sensitivity of gonadotrophs to GnRH, likely to be related to their immature state at the silver stage. The similar efficacy of mGnRH and cGnRH-II suggested that the pituitary GnRH receptor had a low specificity toward various molecular forms, in the eel as in the other nonmammalian species. The protein kinase C (PKC) activator (phorbol ester: PMA) also stimulated GTH secretion, with a maximal effect at 10−8M,indicating that the PKC pathway was functional. In contrast, a depolarizing agent (50 mMKCl) had no significant effect on GTH release, suggesting lack of a functional voltage-sensitive calcium channel (VSCC) secretory pathway. Perifusion experiments on whole pituitary confirmed the lack of effect of KCl on gonadotrophs from E2-pretreated eels and indicated that an additionalin vivotreatment with GnRH agonist and dopamine antagonist could induce the differentiation of a functional VSCC pathway. These characteristics of the transduction mechanisms may be related to the immature state of the eel gonadotrophs at the silver stage.