Studies on chemosensory communication generally rely on using samples of emanations that have been frozen for convenience, and assume that freezing has no detrimental effects. Female urine triggers courtship ultrasonic vocalizations (USVs) in male mice, and in this study, we examined whether freezing urine affects males' USV responses. We used wild-derived house mice (Mus musculus musculus), and recorded males' USVs after being presented samples of fresh versus frozen urine. We found that males emitted significantly fewer USVs when presented with frozen versus fresh urine, and furthermore, males were no longer able to discriminate the scent of familiar versus unfamiliar females after samples were frozen. Our findings indicate that freezing alters the bioactivity of urinary compounds both by reducing its potential to elicit behavioral responses from receivers (male courtship USVs) and eliminating important information about senders (familiarity). Freezing may denature the major urinary proteins (MUPs) released in rodent urine that play an important role in chemical communication. Our results raise concerns about the negative results of studies utilizing frozen samples for studying chemical signals.