Long-term exposure to antivirals is associated with serious cellular toxicity to the kidney. Organic Anion Transporters (Oats) are believed to mediate the cellular uptakeand hence cytotoxicity, of many antivirals. However, a systematic in vitro and ex vivo analysis of interactions between these compounds with various Oat isoforms has been lacking. To characterize substrate interactions with mOat1, mOat3, and mOat6, a fluorescence-based competition assay in Xenopus oocytes as well as wildtype and knockout whole embryonic kidney (WEK) organ culture systems was developed using 6-carboxyfluorescein, 5-carboxyfluorescein, and fluorescein. Of nine common antiviral drugs assessed in oocytes, many manifested higher affinity for SLC22a6 (mOat1), originally identified as NKT (eg. adefovir and cidofovir), two (ddC and ddI) manifested significantly higher affinity for mOat3, while mOat6 possessed comparatively low but measurable affinity for certain antivirals. A live organ staining approach combined with fluorescent uptake in WEK cultures allowed the visualization of Oat-mediated uptake ex vivo into developing proximal tubule-like structures, as well as quantification of substrate interactions of individual Oat isoforms. In general, antiviral specificity of SLC22a6 (Oat1) (in Oat3-/- WEK culture) and of SLC22a8 (Oat3) (in Oat1-/- WEK culture) was consistent with the Xenopus oocyte data. The combined observations suggest SLC22a8 (Oat3) is the major transporter interacting with ddC and ddI. Finally, Quantitative Structure-Activity Relationship (QSAR) analysis of the nine antivirals’ physicochemical descriptors with their Oat affinity indicates that antiviral preferences of mOat1 are explained by high polar surface areas (eg. phosphate groups), whereas mOat3 prefers hydrogen bond acceptors (eg. amines, ketones) and low rotatable bond numbers. In contrast, hydrogen bond donors (eg. amides, alcohols) diminish binding to mOat6. Taken together, the data provide a basis for understanding potential drug interactions in combination antiviral therapy, as well as suggesting structural modifications to enhance or diminish these effects through drug design.