The secondary structure of bovine serum albumin (BSA) in the binary surfactant system of anionic sodium dodecyl sulfate (SDS) and zwitterionic N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DDAPS) was examined at 25°C. The helicity of BSA decreased from 66% to 55% in a solution of DDAPS alone and decreased to 50% in a solution of SDS alone. However, the late addition of DDAPS reformed the helical structure of BSA, which was initially disrupted by SDS. The reformation required higher DDAPS concentrations as the initial SDS concentration increased. A maximum helicity of 63% was attained by this reformation. On the other hand, the helical structure of the protein, which was first affected by DDAPS denaturation, was also reformed to the same degree by the late addition of certain amounts of SDS. Although attention was paid to the additive order of these two surfactants to BSA, the final helicity of the protein depended on the final concentrations of these two surfactants, irrespective of the additive order. These phenomena may be attributed to the predominance of mixed micelle formation over complex formation between BSA and the two surfactants below the mixing ratio of DDAPS ([DDAPS]/([DDAPS]+[SDS])) of 0.95. The predominance of the mixed micelle formation distinctly appeared in mixing ratios between 0.50 and 0.75. In this range, the mixed micelle formation accompanied the removal of dodecyl sulfate (DS) ions bound to BSA upon the late addition of DDAPS to the BSA-SDS mixture, whereas, upon the late addition of SDS to the BSA-DDAPS mixture, the mixed micelle formation was accelerated by the coexistence of DDAPS which disturbed the binding of DS ions to the protein.