Streptolysin O (SLO), a polypeptide of 571 amino acids, belongs to a family of highly homologous toxins that bind to cell membranes containing cholesterol and then polymerize to form large transmembrane pores. A conserved region close to the C terminus contains the single cysteine residue of SLO and has been implicated in membrane binding, which has been the only clear assignment of function to a part of the sequence. We have used a cysteine-less active mutant of SLO to introduce single cysteine residues at 19 positions distributed throughout the sequence. The cysteines were derivatized with the polarity-sensitive fluorophore acrylodan, and the fluorescence emission of the label was examined at the different stages of SLO pore assembly. With several mutants, oligomerization on membranes was accompanied by emission blue-shifts, indicating movement of the label into a more hydrophobic environment. These effects were essentially confined to the range of amino acids 213-305. With oligomeric mutants L274C, S286C, and S305C, additional environmental alterations were induced when different nondenaturing detergents were used to dislodge the membrane lipids from the oligomers. The corresponding amino acid residues thus insert into the lipid bilayer during pore formation. Conversely, the spectra of oligomeric mutants A213C and T245C were not affected by detergents. Devoid of contact with the lipid bilayer, these amino acid residues probably participate in the interaction of SLO molecules within the oligomer.