Colicin E1 was altered by oligonucleotide-directed mutagenesis at the site of three charged residues on the COOH side of the 35-residue hydrophobic segment in the channel-forming domain. Asp-509 is one of five conserved acidic residues in the channel domain of colicins A,B,E1,Ia, and Ib and is the first charged residue following the hydrophobic segment, followed by the basic residues Lys-510 and Lys-512. Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon. Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a nonsuppressing cell strain harboring the mutant plasmid while full-length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gln, Lys-512 to Tyr, were obtained by using appropriate suppressor strains. The truncated colicins displayed (i) a low cytotoxicity, .apprx. 1% of intact wild-type colicin, (ii) 10-fold less in vitro channel activity with lipsomes, and (iii) reduced labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer. (iv) The truncated mutants also displayed an altered conformation at pH 6 that allowed greater binding and activity with liposomes at this pH relative to wild type. The cytotoxicity of single residue substitutions at Aap-509 showed a range of cytotoxicities, wild type > Swe-509 > Gln-509 > Leu-509, although none of these changes greatly affected the in vitro channel activity or pH dependence. Substitution of a neutral residue for either of the positively charged Lys residues in point mutants Lys-510 .times. Met or Lys-512 .times. Tyr also did not significantly affect in vivo or in vitro activity. However, the anion selectivity of the channel was significantly decreased by the latter mutations, indicating that Lys-510 and Lys-512 affect the ion selectivity, probably at the surface of the channel.