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Testosterone concentrations in serum of rats bled throughout pregnancy and post partum were measured using Celite microcolumn chromatography and a radioimmunoassay for testosterone. Mean serum levels of testosterone ranged from about 170 to 340 pmol/l during the first 10 days of pregnancy. Significant increases in concentrations of testosterone in serum of pregnant rats were found on days 12, 15 and 18 of gestation. The highest testosterone concentrations occurred on days 18 and 20 of pregnancy when mean levels were 3228 and 3685 pmol/l respectively. Testosterone levels declined before parturition on day 22 (mean = 1449 pmol/l and declined further after parturition (mean = 315 pmol/l). In order to determine whether serum testosterone concentrations varied during the day in the pregnant rat, samples were collected at 6-h intervals on days 6–7 and 14–15 of gestation. Diurnal variations in serum testosterone concentrations were not evident during early or late pregnancy, unlike the rhythmic changes in serum prolactin levels found at these times during early pregnancy. The possible sources of the increased titres of serum testosterone during the second part of gestation in rats are discussed.