Limbic system functioning is integral to the control and modulation of affect, motivation, reward, and memory. Neuropsychiatric disturbances involving disruptions in these cognitive and emotional dimensions exhibit different prevalence rates for men and women. Gender-specific differences in this integrated brain area may therefore be important in understanding both normal behavioral functioning and the etiologic underpinnings of neuropsychiatric disorders. To further explore such differences in limbic system function, we assessed regional cerebral blood flow, by SPECT, in men and women following the administration of procaine. Procaine is a local anesthetic that preferentially stimulates limbic structures. Psychiatrically and medically healthy, age-matched women (n = 15, 33.2 +/- 6.9 years) and men (n = 15, 32.8 +/- 6.9 years) were administered 1.38 mg/kg procaine or saline intravenously in two separate sessions. Using voxel-based analyses (P < 0.001), males significantly activated the bilateral insular cortex following procaine, whereas females more strongly activated the bilateral anterior and mesial temporal cortex. Both groups demonstrated significant anterior cingulate activation. Subjective responses to procaine did not significantly differ between the men and women. To our knowledge, this is the first report demonstrating gender-specific responses in limbic activation following a pharmacologic challenge. These findings suggest that men and women can activate different limbic structures following the same provocative pharmacologic stimulus, despite sharing a similar subjective experience. Studies assessing pharmacologic challenges of limbic system structures should consider gender as a critical variable in assessing biologic responsiveness.