The effects of testosterone propionate (TP) on brain mechanisms involved in the sexual differentiation of ultrasonic vocalizations were examined in Mongolian gerbils (Meriones unguiculatus). Treatment of neonatal females with TP fully masculinized the rate of emission of the upsweep precopulatory ultrasound during adult sexual interactions with oestrous females. Intracranial implantation of small crystals of TP mixed with cholesterol (65 ng) into females 1–15 h after birth also masculinized the upsweep vocalization emitted in adulthood. Implants of TP positioned in the hypothalamic area had a significantly greater masculinizing effect than TP implants outside this region, or pure cholesterol implants. Two other sexually dimorphic vocalizations, the modulated (mainly precopulatory) and unmodulated (mainly copulatory) calls were masculinized by systemic TP, but intracranial TP had no significant masculinizing action on these calls. Genital structures of females which received neonatal injections of TP were strongly virilized in that their clitorides were lengthened and male-type cornified spines were present on the glans. Females which had received intracranial implants of TP were not virilized peripherally in adulthood. We conclude that testosterone or its metabolites have a direct hypothalamic effect on the development of masculine upsweep vocalizations. Because the other vocalizations were insensitive to intracranial TP, the underlying neural tissues may have different thresholds of response to androgen.