Females demonstrate higher cerebral perfusion compared to males; however, the mechanisms for this difference remain unknown. The role of estrogen has been implicated in the upregulation endothelial nitric oxide synthase (NOS) in animal models and speculated in humans. Therefore, it was hypothesized that females exhibit greater NO contribution to basal cerebral perfusion compared to males. Healthy females (n=6) and males (n=6) underwent quantitative MRI cerebrovascular assessment during intravenous infusion of saline and the NOS inhibitor L-NMMA (5mg/kg/min) in a randomized, single-blind design. Pseudo-continuous arterial spin labeling magnetic resonance imaging (MRI) was used to quantify microvascular perfusion which was normalized to gray matter volume. Heart rate, mean arterial pressure, and end-tidal CO2 were measured during study procedures. Data were analyzed via two-way repeated measures ANOVA (Sex x Condition) and by unpaired t-test as appropriate. Data are mean ± SD and significance was set at p<0.05. Females and males did not differ in age (23±2 vs. 22±2 years, respectively; p>0.99) or BMI (22±1 vs. 22±2 kg/m2, respectively; p=0.85). There were no differences between groups or conditions for heart rate (Females 59±4 vs. Males 56±7 bpm, sex p=0.29; Saline 58±5 vs. L-NMMA 57±7 bpm, condition p=0.77), mean arterial pressure (Females 85±6 vs Males 85±6 mmHg, sex p=0.91; Saline 86±7 vs 84±4 mmHg, condition p=0.42),and end-tidal CO2 (Females 35±2 vs. Males 37±3 mmHg, sex p=0.16; Saline 36±3 vs. L-NMMA 36±2 mmHg, condition p=0.65). Gray matter volume did not differ between sexes (Females 790±49 vs Males 830±98 mL; p=0.34). Females displayed approximately 20% greater global cerebral perfusion compared to males (43±6 vs 35±4 mL/100g/min, respectively; sex p=0.01). L-NMMA reduced perfusion compared to saline infusion (Saline 41±5 vs LNMMA 38±8 mL/100g/min; condition p=0.02). The NOS-mediated change in perfusion did not differ between sexes (Females: -10±12 vs Males: -4±5%, respectively; p=0.145). Regional analyses of the frontal lobe, parietal lobe, temporal lobe, occipital lobe, and cerebellum revealed similar patterns of sex differences and L-NMMA effects. These data support previous findings that females exhibit greater cerebral perfusion compared to males. Additionally, NO appears to contribute to basal cerebral perfusion. These data also suggest NO-independent pathways are likely to exist that may explain sex differences in basal cerebral perfusion.