Four experiments were conducted to determine the chemosensory modality that supports ultrasonic courtship vocalizations by male mice to females and to chemosignals from females. Both removal of the olfactory bulbs (Experiment 1) and removal of the vomeronasal organ (Experiments 2-4) produced similar deficits in the pattern of ultrasonic vocalizations elicited by conspecifics or their odors. Removal of the vomeronasal organ did not impair the ability to locate food buried under cage shavings. These results are consistent with the notion that the analysis of food related odors is subserved by olfaction and that vocalizations to sex chemosignals are elicited primarily by stimulation of the vomeronasal organ/accessory olfactory bulb. Removal of the vomeronasal organ did not induce seminal vesicle regression or lower plasma immunoreactive testosterone levels (Experiment 2) nor was an attempt to restore vocalizations with exogenous testosterone successful (Experiment 4). Thus the altered vocalization pattern following removal of the vomeronasal organ does not appear to arise as a motivational deficit mediated by androgens. Experiments 2 and 3 demonstrated that, in the absence of the vomeronasal organ, stimulation of other sensory systems can, to some extent, maintain the male's tendency to vocalize more to females or their odors than to males or their odors. However, this responsiveness to females may rely upon additional behavioral cues. Previous experience also plays a considerable role in the response to chemosensory gender cues by males who lack their vomeronasal organs. Removal of the vomeronasal organ prior to adult heterosexual encounters (Experiment 3) virtually eliminated the male's responsiveness to either anesthetized females or their chemosignals. Hence males require adult heterosexual experience with a functioning vomeronasal organ before other chemosensory systems acquire the ability to mediate gender recognition as measured by ultrasonic vocalizations.