The emission of ultrasonic vocalization (USV) by rats submitted to the formalin test has not yet been demonstrated. We performed two experiments to establish the formalin concentration to induce USV and the relationship of USV emission with motor behaviors and the effects of morphine and naloxone on USV during the formalin test. Male Wistar rats were used. In Experiment 1, 3 different groups of rats were subcutaneously injected with 5%, 10%, or 12.5% formalin in 1 of the anterior paws. Experiment 2 was intended to verify the effect of morphine 1, 2.5, or 5 mg/kg on USV during the 12.5% formalin test, whereas other groups of rats received naloxone 2 mg/kg with each one of the morphine doses to verify the specificity of opioid action. USV and motor behaviors were simultaneously measured in 5-min windows for 40 min, and early (0-5 min), interphase (5-20 min), and late (20-40 min) phases of the test were characterized. Vocalization was detected mostly during the interphase of the formalin test, mainly after formalin 12.5%. Morphine suppressed USV in a naloxone-reversible manner. This is a demonstration of USV during the formalin test, allowing the inclusion of an additional nonreflex behavioral measure to help characterize more clinically relevant integrated behavioral patterns in this rat model of pain.