Organic Anion Transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison to the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over forty structurally diverse organic anions, we find a correlation between organic anion potency (pKi) and hydrophobicity (LogP) suggesting a hydrophobicity-driven association with transporter binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative Molecular Field Analysis (CoMFA) confirms these mechanistic insights and provides a model for predicting new OAT substrates. By CoMFA, both hydrophobic and charged interactions contribute to Oat1 binding, whereas it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the 3-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (Vmax) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity to a particular transporter may enable design of drugs for targeting to specific tissues (e.g., olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g., kidney, olfactory mucosa) via organic anions.