Purpose Studies with humans have shown that acute opioid administration increases aggressive behavior, but no laboratory study has yet investigated whether opioid withdrawal produces comparable effects. Methods This study investigated whether mild opioid deprivation affected aggression in 6 males and 6 females (N = 12) undergoing methadone maintenance therapy. Aggressive behavior was measured using the point subtraction aggression paradigm (PSAP). Trait aggression was assessed using the Buss-Perry Aggression Questionnaire. Behavior on the PSAP and mood ratings on the Profile of Mood States questionnaire (POMS) were measured on two separate days; once before and once following the participant's daily methadone dose, with order counterbalanced across participants. Results There were no effects of opioid withdrawal on aggressive responding, control response rates, monetary reinforced responding, or self-reports of mood. Rates of responding on the monetary-reinforced option increased across sessions regardless of condition, suggesting an exposure effect. Conclusion These data suggest that mild opioid deprivation occurring 24 hours following methadone administration in individuals undergoing methadone maintenance therapy does not increase aggression, at least in individuals showing low levels of aggressive behavior and low measures of trait aggression. Because participants reported no mood changes and there were no direct measures of withdrawal, it is possible that 24 hours of methadone deprivation produced no withdrawal in these participants. It remains to be determined whether more prolonged deprivation might generate more significant changes in mood and/or changes in aggressive behavior.