Developmental effects of phytoestrogens were studied in offspring from pregnant rats who received a free-feeding diet of either rat chow that was very low in phytoestrogens (low phyto), rat chow low in phytoestrogens and given a genistein and diadzein supplement tablet (high phyto), or normal rat chow (normal) from the second week of pregnancy to weaning (postnatal day 21). Measurements of anogenital distance, daily weights, righting reflex and ultrasonic vocalizations were made on neonatal pups and plasma testosterone and corticosterone were assessed in adult males. There was a significant effect of phytoestrogen treatment on USV for all male and female offspring. Differences between groups in daily weights and anogenital distance were attributed to the micronutrient levels of the two rat chow types employed in this study. No differences in righting reflex test, corticosterone levels or testosterone levels were found among treatment conditions. These results are the first demonstration of phytoestrogens affecting USVs and underscore the complexity of the effects of these substances on biobehavioral development.