There are several lipid binding sites on serum albumins. The aim of this study was to examine the binding of bovine serum albumin (BSA) to cholesterol (Chol), 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP), (dioctadecyldimethyl)ammonium bromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE), at physiological conditions, using constant protein concentration and various lipid contents. Fourier transform infrared (FTIR), circular dichroism (CD) and fluorescence spectroscopic methods were used to analyze the lipid binding mode, the binding constant, and the effects of lipid complexation on BSA stability and conformation. Structural analysis showed that lipids bind BSA via both hydrophilic and hydrophobic contacts with overall binding constants of K-Chol = (1.12 +/- 0.40) x 10(3) M-1, K-DDAB = (1.50 +/- 0.50) x 10(3) M-1, K-DOTAP = (2.45 +/-0.80) x 10(3) M-1, and K-DOPE = (1.35 +/- 0.60) x 10(3) M-1. The numbers of bound lipid (n) were 1.1 (cholesterol), 1.28 (DDAB), 1.02 (DOPE), and 1.21 (DOTAP) in these lipid-BSA complexes. DDAB and DOTAP induced major alterations of BSA conformation, causing a partial protein unfolding, while cholesterol and DOPE stabilized protein secondary structure.