Human MATE1 (Multidrug And Toxin Extrusion 1, hMATE1) is an electroneutral H+/organic cation (OC) exchanger mainly expressed in the apical membrane of renal proximal tubule cells. It plays a critical role in the final step of organic cation excretion of the kidney, and these OCs comprise about 40% of prescribed drugs. Understanding the transport turnover rate (TOR) has important consequences for investigation of the transport mechanism, and is critical for interpreting in vitro‐in vivo extrapolation (IVIVE) required for physiologically‐based pharmacokinetic (PBPK) modeling to predict the influence of transport on pharmacokinetics. In the present study, we purified MATE1 protein from CHO cells stably expressing V‐5 epitope tagged hMATE1 by cell surface biotinylation, and the amount of MATE1 protein was obtained by quantitative western analysis using standard curves based on the V‐5‐containing Multiple tag fusion protein (GenScript). Cell surface protein represented approximately 25% of total MATE1 protein expressed by the cell. The number of cell surface transporters was 29.2 fmol cm−2. (~50,000 transporters per cell). The kinetics of MATE1‐mediated uptake of 1‐methyl‐4‐phenylpyridinium (MPP), atenolol, and metformin were performed at pH 7.4 (room temperature) in these CHO cells following an ammonia pulse to produce an outwardly‐directed H+ gradient. Apparent Kt and Jmax values (±SE; n=4) were: MPP, 79.6 ± 10.9 μM and 284 ± 65 pmol cm−2 min−1; atenolol, 352 ± 67 μM and 398 ± 121 pmol cm−2 min−1; metformin, 629 ± 60 μM and 1,561± 153 pmol cm−2 min−1. TOR values (±SE, n=4) calculated using the number of transporters and Jmax values were: MPP, 180 ± 41 s−1; atenolol, 253 ± 77 s−1; and metformin, 993 ± 97 s−1. These values are consistent with TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease) and uniporters (GLUTs, ENTs).