Background and Purpose— Women experience worse outcomes after stroke compared with men. Prior work has suggested sex-based differences in coagulation and fibrinolysis markers in subjects with acute stroke. We explored whether sex might modify the effect of recombinant tissue plasminogen activator (rtPA) on outcomes in patients with acute ischemic stroke. Methods— Using a combined database including subjects from the National Institute of Neurological Disorders and Stroke (NINDS), Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke (ATLANTIS) A and B, and the Second European Cooperative Acute Stroke Study (ECASS II) trials, we examined 90-day outcomes in patients randomized to rtPA versus placebo by sex. We used logistic regression to control for potential confounders. Results— Among 988 women treated between 0 and 6 hours from symptom onset, patients receiving rtPA were significantly more likely than those receiving placebo to have a modified Rankin Score ≤1 (40.5% versus 30.3%, P <0.0008). Among 1190 men, the trend toward benefit in the overall group did not reach statistical significance (38.5% versus 36.7%, P =0.52). An unadjusted analysis showed that women were significantly more likely to benefit from rtPA compared with men ( P =0.04). Controlling for age, baseline National Institutes of Health Stroke Scale, diabetes, symptom onset to treatment time, prior stroke, systolic blood pressure, extent of hypoattenuation on baseline computed tomography scan and several significant interaction terms (including onset to treatment time–by-treatment and systolic blood pressure–by treatment) did not substantially change the strength of the interaction between gender and rtPA treatment ( P =0.04). Conclusions— In this pooled analysis of rtPA in acute ischemic stroke, women benefited more than men, and the usual gender difference in outcome favoring men was not observed in the thrombolytic therapy group. For patients presenting at later time intervals, when the risks and benefits of rtPA are more finely balanced, sex may be an important variable to consider for patient selection.