Abstract: We previously reported that the psychostimulant drug methcathinone inhibits serotonin accumulation via the plasma membrane serotonin uptake transporter. By analogy to known substrates for the serotonin transporter, we hypothesized that methcathinone is also a substrate for this transporter and that inhibition of serotonin uptake by methcathinone occurs in part through competition for substrate recognition sites within the transporter. To test the hypothesis we preloaded human platelets with [3H]5-HT then superfused the platelets with either methcathinone or with the known serotonin uptake transporter substrate para-methylthioamphetamine. Under superfusion conditions, transporter substrates will evoke an increase in released [3H]5-HT through a carrier-mediated exchange process. For direct assessment of methcathinone transport via the serotonin uptake transporter, we tested whether [3H]methcathinone would be accumulated by cells stably expressing the cloned human serotonin uptake transporter (293SERT cells). Supporting the hypothesis, superfusion of [3H]5-HT-containing platelets with methcathinone or with para-methylthioamphetamine produced a large increase in tritium efflux. The efflux declined when the drugs were removed. When increasing concentrations of [3H]methcathinone were incubated with 293SERT cells under conditions used to assess serotonin transport, saturable, single-site accumulation of radiolabel was observed. The uptake of [3H]methcathinone was temperature, inhibitor, and sodium-sensitive, and was not observed in wild-type HEK 293 cells. Non-linear regression analysis of specific [3H]methcathinone uptake produced values for KM and Vmax of 244±51 nM and 202±25 fmol/min./mg protein, respectively. These data support the notion that the reported serotonergic neurotoxicity of methcathinone may arise through accumulation of the drug within serotonergic neurones.