Alkylphenols, considered to be endocrine disruptor chemicals and toxic environmental priority pollutants, pose great threats to humans with wide exposure from food and other potential sources. In this paper, to evaluate the toxicity of the alkylphenols at the protein level, the effects of 4-tert-octylphenol (OP) and 4-nonylphenol (NP) on human serum albumin (HSA) were characterized by molecular modeling, steady state and time-resolved fluorescence, ultraviolet-visible spectroscopy (UV-vis) and circular dichroism spectroscopy (CD). The enthalpy change (Delta H) and entropy change (Delta S) indicated that hydrophobic forces and hydrogen bonds were the dominant intermolecular forces in the binding of the alkylphenols to HSA. The binding constant of HSA-NP is much greater than that of HSA-OP, revealing that NP, which has a longer carbon chain, has a higher affinity than OP. The alterations of protein secondary structure in the presence of the alkylphenols were confirmed by UV-vis and CD spectroscopy. The time-resolved fluorescence study showed that the lifetimes of tryptophan (Trp) residue of HSA decreased after the addition of the alkylphenols, NP with longer carbon chain impact on the average lifetime of the Trp of HSA more than OP, which consistent with the conclusion drawn from the fluorescence date. (C) 2013 Elsevier B.V. All rights reserved.