Organic cation transporter 1 (OCT1) is located in the sinusoidal membrane of human hepatocytes. It mediates the uptake of hydrophilic organic cationic drugs in hepatocytes and thus determine their systemic concentrations. OCT1 has a broad spectrum of structurally diverse substrates like metformin, sumatriptan, trospium, and fenoterol. Recent cryo-EM data suggested that Y361, E386, and R439, referred to as the YER motif, could be important for transport. Building on this, we used extensive functional analyses to investigate the general function, and the substrate-specific effects of the YER motif. We determined the activity of the Y361A, E386A, and R439A mutants for fifteen OCT1 substrates. Extended mutagenesis revealed the negative charge of E386 and the positive charge of R439 as essential for the transport of all substrates tested. Charge reversal mutants, E386R-R439E, did not restore transport activity, suggesting that at least one of the two amino acids is involved in additional interactions essential for transport. Y361 exhibited substrate-specific effects. The Y361A mutant transported fenoterol, but not pirbuterol or other beta