Organic anion and cation transporters (OATs, OCTs, and OCTNs) mediate the proximal tubular secretion of numerous clinically important compounds, including various commonly prescribed pharmaceuticals. Here, we report determination of the ontogeny of these transporters and of NaP(i)2 and SGLT1, using quantitative polymerase chain reaction (QPCR) to determine expression levels of transporter genes in rat embryonic kidneys on each day of gestation from embryonic day (ed) 13 to ed18, in cultures of induced and uninduced metanephric mesenchyme (MM), and on each day of 1 week of whole embryonic kidney (WEK) culture. We also examined ontogeny of Oat1 protein expression in rat embryonic kidney by immunohistochemistry. Finally, we used uptake of fluorescein (FL) as a novel in vitro functional assay of OAT expression in WEK and MM. Developmental induction of OAT and OCT genes does not occur uniformly: some genes are induced early (e.g., Oat1 and Oat3, potential early markers of proximal tubulogenesis), and others after kidney development is relatively advanced (e.g., Oct1, a potential marker of terminal differentiation). The ontogeny of transporter genes in WEK and MM is similar to that observed in vivo, indicating that these organ culture systems may represent convenient in vitro models to study the developmental induction of OATs, OCTs, and OCTNs. Functional transport was evidenced by accumulation of FL in the developing tubule in WEK and MM organ cultures. Our findings on the renal ontogeny of OATs and OCTs could carry implications both for the development of more rational therapeutics for premature infants, as well as for our understanding of proximal tubule differentiation.