Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in ⁴⁵Ca²⁺ and cAMP accumulation. Cortisol's early actions occur through a protein-synthesis-independent pathway and are mimicked by a membrane impermeable analogue. The signaling pathway that mediates rapid, non-genomic, membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca²⁺ _i) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca²⁺-sensitive dye, fura2, revealed that cortisol reversibly reduces basal and hyposmotically-induced Ca²⁺_i within seconds (P< 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca²⁺_i. Under depolarizing [K⁺], the L-type calcium channel agonist BAY-K-8644, a factor known to delay the closing of L-type Ca²⁺ channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY-K-8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca²⁺ that likely involves an attenuation of Ca²⁺ entry through L-type voltage-gated Ca²⁺ channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.