Testosterone, estradiol, and progesterone levels were measured in the second trimester of pregnancy in maternal serum and amniotic fluid, and related to direct observations of gender-related play behavior in 63 male and 63 female offspring at age 13 months. During a structured play session, sex differences in toy preference were found: boys played more with masculine toys than girls (d = .53) and girls played more with feminine toys than boys (d = .35). Normal within-sex variation in prenatal testosterone and estradiol levels was not significantly related to preference for masculine or feminine toys. For progesterone, an unexpected significant positive relationship was found in boys between the level in amniotic fluid and masculine toy preference. The mechanism explaining this relationship is presently not clear, and the finding may be a spurious one. The results of this study may indicate that a hormonal basis for the development of sex-typed toy preferences may manifest itself only after toddlerhood. It may also be that the effect size of this relationship is so small that it should be investigated with more sensitive measures or in larger populations.