Abstract Plasma testosterone levels were higher in pooled samples from male fetuses than from female fetuses on day 19 of pregnancy. Plasma testosterone from female fetuses with males located caudally in the uterus was higher than from females that lacked such males. Testosterone level of both male and female fetuses was correlated with maternal testosterone. No correlation was found between maternal testosterone and number of males in the litter, male-to-female ratio, or litter size. These results corroborate earlier findings of a sex difference in plasma testosterone levels on fetal day 19 in rats, and provide support for the hypothesis that female rats receive androgens from males located caudally in the uterus. No evidence was found that testosterone of pregnant females is affected by the sex ratio or size of her litter.