Ongoing HIV replication within gut lymphoid tissues may contribute to the persistence of HIV despite treatment with antiretrovirals (ARVs). ARVs may have reduced exposure in certain tissue areas, but current methods for assessing ARV tissue concentrations cannot test this hypothesis. The goal of this project was to characterize how ARVs distribute within gut tissue, and determine whether or not they concentrate in areas of local HIV gene expression. Drug transporter expression and localization were also evaluated in these tissues to determine what factors influence ARV distribution. Using mass spectrometry imaging (MSI), the ileum and rectum of humanized mice (n=49), non-human primates (NHP, n=12) and humans (n=5) were evaluated for ARV distribution. The co-localization of ARV distribution with CD3+ T cells, drug efflux transporters, and HIV RNA expression was assessed. ARV correlation with CD3+ T cells ranged from -0.09 to 0.32 and was not significantly different between species. HIV RNA was not co-localized with ARV exposure in any species (r range -0.09-0.2). ARV-transporter co-localization was highest for MDR1 in all species, and not significantly different between the ileum and rectum. MSI provided previously unobtainable distributional data, showing ARV localization to specific tissue sites and no co-localization with HIV gene expression. Drug transporters affect ARV tissue disposition and can be exploited to maximize ARV exposure, but quantitative measures of drug transporter protein expression across preclinical species are not available. Gene and protein expression of ARV efflux and uptake transporters were evaluated using qPCR, Western blot, and LC-MS proteomics. Gene and protein expression were generally consistent between infected and uninfected animals and between ileum and rectum. There was poor correlation between methods, and no single method significantly predicted tissue ARV concentrations in a stepwise regression model. We also show that the contribution of human transporter isoforms in humanized mice can significantly affect interspecies comparisons. Human protein expression data was most consistent with humanized mice (1-9 fold different) over NHPs (1-21 fold different). By completing these experiments in two animal species and in humans, we can better understand how HIV persists in tissues and inform the development of targeted therapies for HIV eradication.