Bacteriophage  uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis.  S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from  S_C51S were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S_C51S (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S_A52V mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S_A52V/C51S is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S_A52V. Both S_A52V (dominant negative) and S_A48V (antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S_A52V and S_A48V, respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.