Ultrasonic vocalizations (USVs) have recently emerged as an indicator of the emotional state of rats, and the evaluation of the USVs in the 50-kHz range has been proposed as a tool to investigate the affective properties of drugs of abuse. To clarify the relevance of 50-kHz USVs to drug-induced reward, the acute and long-term effects elicited by different psychoactive drugs [ amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), methylphenidate, morphine, and nicotine ] were characterized in adult male rats. Amphetamine and methylphenidate were the only drugs that stimulated the emission of 50-kHz USVs by rats after their acute administration. Moreover, amphetamine was the only drug that elicited a significant emission of 50-kHz USVs after repeated administration. However, rats in all the treatment groups emitted 50-kHz USVs when later re-exposed to the environment previously paired with repeated drug administration, likely indicative of drug-mediated environmental conditioning. Taken together, these results demonstrate the existence of major differences in the acute and long-term effects of different psychoactive drugs on the emission of 50-kHz USVs by rats. Moreover, these results provide a better understanding of the usefulness of 50-kHz USVs as a new tool for the assessment of drug-mediated reward, with implications for the preclinical study of addictive behaviors