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Three-dimensional, organ-on-chip models that recapitulate kidney tissue are needed for drug screening and disease modeling. Here, we report a method for creating a perfusable 3D proximal tubule model composed of epithelial cells isolated from kidney organoids matured under static conditions. These organoid-derived proximal tubule epithelial cells (OPTECs) are seeded in cylindrical channels fully embedded within an extracellular matrix, where they form a confluent monolayer. A second perfusable channel is placed adjacent to each proximal tubule within these reusable multiplexed chips to mimic basolateral drug transport and uptake. Our 3D OPTEC-on-chip model exhibits significant upregulation of organic cation (OCT2) and organic anion (OAT1/3) transporters, which leads to improved drug uptake, compared to control chips based on immortalized proximal tubule epithelial cells. Hence, OPTEC tubules …