From previous research, the ultrasonic vocalizations of male mice (Mus domesticus) to female mouse urine were hypothesized to be learned as a result of classical conditioning during adult heterosexual encounters. According to this interpretation, a previously neutral conditioned stimulus in female urine comes to elicit vocalizations as a result of its association with some other unknown unconditioned stimulus associated with adult females. However, the research from which this hypothesis was derived utilized urine collected from females housed in metabolic cages. Three experiments further examined the classical conditioning hypothesis using two types of female urine: (i) metabolic-cage-collected urine and (ii) freshly voided urine. Experiment 1 demonstrated that, in contrast to vocalizations to metabolic-cage-collected urine, adult heterosexual experience was not necessary for males to vocalize to freshly voided female urine. In addition, unlike metabolic-cage-collected urine (Experiment 3), freshly voided urine remained a potent stimulus for eliciting vocalizations during repeated testing (Experiments 2 and 3). Finally, freshly voided urine appeared to cause a previously neutral stimulus (cotton swab) to acquire ultrasound eliciting properties (Experiment 2). We suggest from these findings that two chemosignals that elicit vocalizations from males may exist in female mouse urine: (i) a potent, but volatile or easily degraded, unconditioned stimulus to which males vocalize without sexual experience and (ii) a nonvolatile, chemically stable conditioned stimulus.