The functional state of the pituitary-gonadal axis was studied in rats on days 18.5–21.5 of fetal life (f) and on day 4 postpartum by measurements of plasma levels of luteinizing hormone (LH) and testosterone (T). LH was measured using an ultrasensitive immunofiuorometric assay. In addition, male fetuses were castrated and exposed to the antiandrogen flutamide (FL; 100mg/kg BW) or the Leydig cell-specific cytotoxic agent ethylene dimethane sulphonate (EDS, 50 mg/kg BW on 2 days) by injections of the drugs to the mothers. Besides LH and T, pituitary levels of LH subunit mRNAs were measured in these animals. The results allowed the following conclusions: (1) the plasma LH levels in both sexes are low (<0.05 µg/1, NIH rLH RP-2) on days f18.5 and fl9.5; (2) a 4- to 5-fold increase in plasma LH occurs between days f19.5 and f20.5, and a 3- to 4-fold sex difference appears (females > males); (3) the activation of fetal testicular T production before day f19.5 takes place in the face of very low plasma LH (<0.02 µg/1), suggesting that some factor(s) other than LH may stimulate the testis at this age; (4) the reciprocal changes of plasma LH and T, and the experiments with castration, EDS and FL demonstrate that testicular feedback regulation of LH secretion is functional from day f19.5 onwards; (5) the parallel changes of T in male and female fetal plasma suggest that T in female fetuses comes from male littermates, and (6) the fetal pituitary-testicular axis is less sensitive to hormonal manipulations than that of the postnatal animal, possibly due to interference of maternal and placental hormones with the pituitary-testicular interactions.