Based on observed changes in the social context for the display of ultrasonic vocalizations, scent marking, aggression, and mounting behavior by male mice with a null mutation of the transient receptor potential 2 ion channel, it was proposed recently that a primary function of the mouse vomeronasal organ (VNO)/accessory olfactory system is sex discrimination. We tested this hypothesis directly by studying the ability of male mice to discriminate between urinary odors of conspecifics of the two sexes and in different endocrine states using habituation-dishabituation tests. Male mice from which the VNO had been surgically removed (VNOx) resembled sham-operated controls (VNOi) in their ability to discriminate between volatile urinary odors from estrous females versus gonadally intact males, as well as between urinary odors from estrous versus ovariectomized females and from gonadally intact versus castrated males. When physical access to stimuli was permitted, VNOi control males strongly preferred to investigate volatile and nonvolatile urinary odorants from estrous females as opposed to intact males, whereas VNOx males showed no such preference. Mating performance in tests with estrous females was equivalent in VNOi and VNOx subjects. Both groups of males preferred to mount an estrous female instead of a castrated male. Our results suggest that the VNO is not required for sex discrimination but instead detects the nonvolatile components of opposite-sex urine that may be used to help prolong contact with individuals that produce these chemosignals.