For this study, purified immature porcine Leydig cells in primary culture were used. After 2 days of culture, the cells were incubated with dexamethasone (5 × 10−9, 1 × 10−7 M) for various periods of time (3–45 h). The media were discarded and treatment was repeated with or without the addition of human chorionic gonadotropin (HCG, 10 mIU/mL) for 3 h. Dexamethasone (10−7 M) decreased testosterone production of HCG-treated cells (up to 40%) in a time-dependent fashion while the lower dose was ineffective. The effect of varying doses (10−8 and 10−6 M) of natural glucocorticoids (corticosterone, cortisol) or synthetic glucocorticoids (triamcinolone, triamcinolone acetonide, betamethasone, dexamethasone) and that of a synthetic progestin (R-5020) on cultured Leydig cells was also studied. After 18 h of preincubation, the various synthetic but not the natural steroids nor R-5020, were able to decrease testosterone production of control and HCG-treated cells by 20–40%. Of a number of other hormonal and nonhormonal substances studied at concentrations of 10−9 – 10−5 M, only lysine8-vasopressin at a concentration of 10−6 M was able to inhibit testosterone production by these cells. These results indicate that dexamethasone and other synthetic glucocorticoids, and to a lesser degree lysine8-vasopressin, may exert a direct inhibitory effect on testosterone production by purified porcine immature Leydig cells in vitro.