Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [I(max(choline))] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas I(max(choline)) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I(0.5(choline)) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I(0.5) values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane alpha-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC(50(choline)) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I(0.5(choline)) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I(0.5(choline)) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I(0.5(choline)) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.