Residues Tyr-110 through Gly-115 of serotonin transporter were replaced, one at a time, with cysteine. Of these mutants, only G113C retained full activity for transport, Q111C and N112C retained partial activity, but Y110C, G114C and G115C were inactive. Poor surface expression was at least partly responsible for the lack of transport by G114C and G115C. In membrane preparations, Y110C through G113C all bound a high affinity cocaine analog similarly to the wild type. Treatment with methanethiosulfonate reagents increased the transport activity of Q111C and N112C to essentially wild-type levels but had no measurable effect on the other mutants. The decreased activity of Q111C and N112C resulted from an increase in the K(M) for serotonin that was not accompanied by a decrease in serotonin binding affinity. Superfusion experiments indicated a defect in 5-HT exchange. Modification of the inserted cysteine residues reversed the increase in K(M) and the poor exchange, also with no effect on serotonin affinity. The results suggest that Gln-111 and Asn-112 are not required for substrate binding but participate in subsequent steps in the transport cycle.