Many soluble factors of neural, endocrine, paracrine and autocrine origin are present in the thymus and modulate its function. Long-term effects of sex steroids have! been documented for thymocytes and cells of the thymic microenvironment. In this report we examine rapid actions of progesterone upon aspects of epithelial cell physiology. Progesterone (0.1-10 mu M) was applied to cultured thymulin-secreting thymic epithelial cells (TS-TEC) and changes in transmembrane potential, transmembrane current, intracellular calcium levels and thymulin secretion were assessed. Rapid changes in electrophysiology and intracellular calcium provide evidence for a membrane-bound progesterone receptor in these cells, in addition to classical cytoplasmic receptors. Application of progesterone to TS-TEC caused electrophysiological changes in 56% of cells (n = 40), activating an inward current (-24 +/- 9 pA at 1 mu M, n = 7, p < 0.02) and dose-dependent depolarization (7.1 +/- 1.8 mV at 1 mu M, n = 19, p < 0.01). Intracellular calcium levels, monitored by the ratiometric fluorescent calcium indicator fura-2, increased within seconds of progesterone (1 mu M) application. Progesterone(1 mu M) increased thymulin levels in supernatant, as measured by ELISA, above the levels in the preapplication period (142 +/- 16% of the preapplication period, n = 3, p < 0.02). This effect was reduced in the presence of cobalt chloride which blocks voltage-dependent calcium channels. In addition, TS-IEC in culture were immunoreactive to antibody AG7. This antibody was raised to a membrane-bound antigen involved in calcium influx subsequent to progesterone binding in sperm. thus we suggest that progesterone acts upon many aspects of TS-TEC physiology through both cytoplasmic and membrane-bound receptors.