Adult mice communicate by emitting ultrasonic vocalizations (USVs) during the appetitive phases of sexual behavior. However, little is known about the genes important in controlling call production. Here, we study the induction and regulation of USVs in muscarinic and dopaminergic receptor knockout (KO) mice as well as wild-type controls during sexual behavior. Female mouse urine, but not female rat or human urine, induced USVs in male mice, whereas male urine did not induce USVs in females. Direct contact of males with females is required for eliciting high level of USVs in males. USVs (25 to120 kHz) were emitted only by males, suggesting positive state; however human-audible squeaks were produced only by females, implying negative state during male-female pairing. USVs were divided into flat and frequency-modulated calls. Male USVs often changed from continuous to broken frequency-modulated calls after initiation of mounting. In M2 KO mice, USVs were lost in about 70-80% of the mice, correlating with a loss of sexual interaction. In M5 KO mice, mean USVs were reduced by almost 80% even though sexual interaction was vigorous. In D2 KOs, the duration of USVs was extended by 20%. In M4 KOs, no significant differences were observed. Amphetamine dose-dependently induced USVs in wild-type males (most at 0.5 mg/kg i.p.), but did not elicit USVs in M5 KO or female mice. These studies suggest that M2 and M5 muscarinic receptors are needed for male USV production during male-female interactions, likely via their roles in dopamine activation. These findings are important for the understanding of the neural substrates for positive affect.