1. The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT(3A) and 5-HT(3B)) possess a positively charged lysine residue within the putative channel lining M2 domain (4' position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wildtype (WT) and mutant 5-HT(3A) receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. 2. WT 5-HT(3A) receptors mediated rapidly activating currents in response to 5-HT (10-90 rise time, 103 ms; EC50, 2.34 μM; Hill coefficient, n(H), 2.87). The currents rectified inwardly, reversed in sign at a potential of -9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t(query half), 2.13 s). 3. 5-HT(3A) receptor subunits in which the 4'lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT(3A) receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. 4. Each of the mutants desensitized more slowly than the WT 5-HT(3A) receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. 5. The single-channel conductance of the WT 5-HT(3A) receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4'lysine mutant receptors. Thus it appears unlikely that 4'lysine is exposed to the channel lumen. 6. Mutation of residues immediately adjacent to 4'lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4'lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization.