Abstract Mu-opioid receptor (MOPr) agonists, such as morphine, produce greater antinociception in male compared to female rats. The ventolateral periaqueductal gray (vlPAG) appears to contribute to this sex-difference despite fewer vlPAG output neurons projecting to the rostral ventromedial medulla in male compared to female rats. This greater projection in female rats suggests that non-opioid activation of vlPAG output neurons should produce greater antinociception in female compared to male rats. This hypothesis was tested by comparing the time course and antinociceptive potency of microinjecting MOPr agonists (morphine, DAMGO, fentanyl) and non-opioid compounds (bicuculline, kainic acid) into the vlPAG of female and male rats. Microinjection of morphine or DAMGO produced antinociception that had a slow onset (peak from 15 to 30 min) and long duration (60 min) compared to the antinociception produced following microinjection of fentanyl, bicuculline, or kainic acid (peak effect at 3 min; duration less than 30 min). No sex-differences in the time courses were evident. All five compounds caused a dose-dependent antinociception when microinjected into the vlPAG. Antinociceptive potency was significantly greater in male compared to female rats following microinjection of morphine, DAMGO, and bicuculline, but not following microinjection of fentanyl or kainic acid. In no case did activation of the vlPAG produce greater antinocicepiton in female compared to male rats. These findings demonstrate that the vlPAG can produce comparable antinociception in female and male rats, but antinociception produced by inhibition of GABAergic neurons (whether by morphine or the GABA A receptor antagonist bicuculline) produces greater antinociception in males.