Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle concentration (cmc) declines. Increasing proportions of DDM, which significantly reduce the amount of monomeric sodium dodecyl sulfate (SDS), make the first denaturation step occur at lower concentrations but weaken and eventually remove the second denaturation step. The logarithm of the unfolding rate constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex. This behavior contrasts with the simplicity of unfolding in chemical denaturants and highlights the changing properties of surfactant micelles. We suggest that the transition from spherical to more elongated micelles leads to inhibition of unfolding kinetics, while weaker binding sites may cause a subsequent rise in unfolding rate constants at higher surfactant concentrations. We propose that shifting micellar binding sites on globular proteins such as ACBP, as opposed to the predefined binding sites on membrane protein surfaces, may lead to nonlinear correlations between activation unfolding energies and SDS mole fraction.