In previous studies, the replacement of His35 in the pore-forming protein α-hemolysin (αHL) with Leu, lie, Pro, Arg, Ser, Thr or Cys yielded inactive polypeptides. Here, we show that modification of the inactive single-cysteine mutant αHL-H35C with iodoacetamide, to form H35CamC, generates significant pore-forming activity. The closely related polypeptides H35N and H35Q have, respectively, essentially no activity and greatly reduced activity. The modified residue in H35CamC, S-carboxamidomethylcysteine, mimicks histidine in volume, polarity and hydrogen bonding potential, but is unable to ionize. Unmodified H35C is defective in the final step of pore formation: the conversion of an inactive heptameric membrane-bound assembly intermediate to a structure containing open channels. It is this step in assembly, that is ameliorated in H35CamC.