We located the binding sites of folic acid with milk alpha- and beta-caseins at physiological conditions, using constant protein concentration and various folic acid contents. FTIR, UV-visible, and fluorescence spectroscopic methods as well as molecular modeling were used to analyze folic acid binding sites, the binding constant, and the effect of folic acid interaction on the stability and conformation of caseins. Structural analysis showed that folic acid binds caseins via both hydrophilic and hydrophobic contacts with overall binding constants of K(folic acid-alpha-caseins) = 48(+/- 0.6) x 10(4) M(-1) and K(folic acid-beta-caseins) = 7.0 (+/- 0.9) x 10(4) M(-1). The number of bound acid molecules per protein was 1.5 (+/- 0.4) for alpha-casein and 1.4 (+/- 0.3) for beta-casein complexes. Molecular modeling showed different binding sites for folic acid on alpha- and beta-caseins. The participation of several amino acids in folic acid protein complexes was observed, which was stabilized by hydrogen bonding network and the free binding energy of -7.7 kcal/mol (acid-alpha-casein) and -8.1 kcal/mol (acid-beta-casein). Folic acid complexation altered protein secondary structure by the reduction of alpha-helix from 35% (free alpha-casein) to 33% (acid-complex) and 32% (free beta-casein) to 26% (acid-complex) indicating a partial protein destabilization. Caseins might act as carriers for transportation of folic acid to target molecules.