Many sex differences can be found in expression of aggression and parental nurturing behaviors. These differences are established during fetal development by the action of gonadal steroid hormones, especially the higher levels of androgens experienced by males, which are caused ultimately by the presence of the testis-determining gene Sry on the Y chromosome. Past studies indicated that intra-uterine position has consequences for behavior and reproduction in mice. Female fetuses developing between two male fetuses are 'masculinized' and differ from females developing between the female fetuses. Here, we tested for behavioral differences between females that were or were not exposed to males during fetal development using a novel model system: production of single sex pregnancies. Embryos generated by in vitro fertilization (IVF) were subjected to single blastomere biopsy at 4-cell stage and subsequently sexed via single cell PCR. Sexed embryos from the same IVF cohorts were transferred to surrogate females to generate single sex (female only, F) and mixed sex (female and male, FM) pregnancies. Female progeny from both types of pregnancies were subjected to three-chamber social approach, social proximity task, scent marking, and maternal aggression behavioral tests. We also measured estrus length and reproductive success of these females. Estrus length was similar in females from the two neonatal conditions (F: 7.21±0.52 vs. FM: 7.35±0.52; mean±SEM) and both types of females bred equally well yielding similar litter sizes (F: 6.8±0.7, n=14 vs. FM: 7.5±0.7, n=12), with a similar progeny sex ratio (F: 54% and FM: 48% females). Females from the two neonatal conditions were indistinguishable in social behavior and exhibited normal social responses in the three-chamber social approach and social proximity tests, towards unfamiliar females. When urine was collected from F and FM females and used as a point source in a scent-marking paradigm, exposed males showed a similar distribution and extent of urinary scent marking but tended to engage in higher durations of sniffing the urine from F females (P=0.02). To assess maternal aggression, F and FM females were mated, and three days after giving birth, were tested in a resident-intruder paradigm. F females exhibited enhancement of pup grooming (P=0.04) and an overall decrease of non-pup activity (P=0.01) prior to male intruder introduction. After introduction of the male intruder F females were more defensive evidenced by higher rates of burying, open-mouth threat/lunges, and attacks towards the male (P=0.006), and decreased latency to display these defensive behaviors (P=0.02). To summarize, our results suggest that females devoid of male exposure during fetal development have reproductive abilities similar to those of females from mixed sex pregnancies, and have normal social interactions with other females. However, they exhibit hyper-maternal behavior both in terms of the care and defense of pups in front of a male intruder, and potentially produce a pheromonal milieu that renders them more attractive to males during olfactory investigations. Further research will focus on investigation of the physiological systems that underlie increase in maternal defensiveness in those females. (platform)