BACKGROUND Cell-associated human immunodeficiency virus (HIV) has been found in tissues whose architecture can limit antiretroviral (ARV) drug penetration, including the genital tract. METHODS In healthy and simian-human immunodeficiency virus-infected rhesus macaques dosed for 10 days with 4-drug combination therapy, we evaluated the spatial distribution of 6 ARV drugs within the male and female genital tract by means of mass spectrometric imaging (MSI). We also measured drug transporter gene expression in these tissues to determine their influence of in situ variability of ARV exposure. RESULTS Through MSI, drug-dependent, heterogeneous ARV accumulation was observed, with preferential accumulation in capsular and epithelial spaces for male and female genital tract tissues, respectively. ARVs were primarily detected as single drug exposure across genital tract tissue sections, with the proportion of tissue where any single ARV was detected (median [range], 58% [5.1%-82.3%] for the vagina and 16.6% [3.9%-88.4%] for the testis) exceeding the detection of any 2 (13.8% [0.2%-30.8%] and 0.9% [0.0%-72.8%], respectively) or any 3 (1.8% [0.0%-2.5%] and 0.0% [0.0%-43.1%]) colocalized ARVs. Most (59.7% [range, 45.8%-90.1%]) of the ARV response in vaginal tissue was found to be colocalized with the blood marker heme, suggesting that its origin was from the vasculature rather than parenchymal tissue, while interindividual variability in ARV penetration was higher in testicular tissue. CONCLUSIONS In both female and male genital tract tissues, ARV penetration did not follow simple trends based solely on molecular size or degree of protein binding, and the combination of MSI and drug transporter expression suggests that multiple mechanisms, including drug transporters, participate in determining local accumulation within this tissue.