The helicity of bovine serum albumin (BSA) decreased both in the reverse micelle of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and in the aqueous solution of the same surfactant (the saturated binding amount to the protein was 70 mol/mol). In both cases, the final helicity was approximately identical to that attained in the aqueous solution of sodium dodecyl sulfate. The fluorescence lifetime of N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to BSA shortened upon incorporation into the reverse micelle. Although the helicity remained unchanged regardless of the change in water content (Rw) in the reverse micelle, the lifetime further shortened with an increase in Rw and then flattened out beyond Rw 22. A similar shortening of the lifetime was observed in the aqueous AOT solution. In the present study, it appeared likely that secondary structural changes of proteins in reverse micelles of ionic surfactants generally resemble those observed in aqueous solutions of the identically charged ionic surfactants. This is probably because an electrostatic interaction between the protein and the surfactant is of considerable importance to the protein structure. In the present system, the essentially same interaction of BSA with AOT might be expected to occur between the protein and the inner-charged double-layer of the reverse micelle, as occurs in the aqueous solution. However, the interaction was not very dependent on Rw in the case of BSA.