Ultrasonic vocalizations (USVs) are emitted in response to a sexual partner before, during, and after copulation. These vocalizations are the so-called 50-kHz USVs and can be subdivided into flat and frequency-modulated (FM) 50-kHz trill calls. In the present series of experiments, the potential unconditioned and conditioned incentive properties of female 50-kHz USVs for male rats were examined. USVs were recorded from sexually receptive females during the precopulatory phase. A complete 10-min song, or single flat or FM trill calls were selected as auditory stimuli for Experiments 1 and 2. As FM trill calls, a multistep call was used in Experiments 1 and 2a, and an upward ramp call was used in Experiment 2b. The auditory stimuli were played back with a loudspeaker to naïve and sexually experienced male rats in a sexual incentive motivation test. The odor of a sexually receptive female rat was also used as an incentive stimulus for comparison. In a third experiment, a devocalized female, a sham female and a male rat were used as incentive stimuli. It was found that the auditory stimuli did not induce approach behavior in naïve and sexually experienced male rats, but the olfactory stimulus did. In addition, the males spent equal amounts of time in the vicinity of devocalized and vocalizing females. These data show that 50-kHz USVs neither are unconditioned nor conditioned incentives for male rats.