Sexually and parentally experienced prairie voles display robust biparental care of pups that is similar between the sexes. Little is known, however, about possible sex differences in the parental behaviours of sexually inexperienced prairie voles. Parental behaviour of adult virgin male and female prairie voles was examined in sham-operated and gonadectomized subjects treated with vehicle or oestradiol. Since arginine-vasopressin (AVP) has been suggested to stimulate parental behaviour in sexually inexperienced males, neural AVP immunoreactivity (AVP IR) was quantified. Most sham-operated and castrated males displayed high levels of parental behaviour (9/9 controls, 6/9 castrates) during a 15-min exposure to pups 4 weeks after surgery, and few behavioural differences were seen between groups. Conversely, almost all gonadally intact (8/9) and gonadectomized (8/9) females attacked pups. Implantation of a 0.1-mg pellet of oestradiol immediately after gonadectomy had little effect on males (9/9 parental), whereas most (5/9) oestradiol-treated females acted maternally. AVP-immunoreactive (AVP-ir) fibre density in the lateral septum (LS) and lateral habenula (LHb), expressed by the number of pixels that covered AVP-ir fibres during computerized optical density analysis, was greater in males than females, was non-significantly reduced in castrated males, and doubled in the LS of oestradiol-treated females. In a second experiment, males tested 8 weeks after similar manipulations remained highly parental though castrated males had almost no AVP-ir fibres in the LS and LHb. Levels of AVP IR in males treated with oestradiol were similar to those observed in intact males. A dramatic sex difference therefore exists in the parental behaviour of adult sexually naive prairie voles which cannot be explained by sex differences in gonadal hormones. Because both castrated and intact males were highly parental, even though castrates had virtually no AVP-ir in the LS or LHb, AVP does not appear to be crucial for their responsiveness toward pups.