The immunoregulatory proteins osteopontin (OPN) and lactoferrin (LF) are both highly abundant in milk, with a conserved ratio between different mammalian species, suggesting that the role of each protein in infant development may be linked. In this study we used isothermal titration calorimetry and differential scanning calorimetry experiments to demonstrate that LF and OPN interact with each other through a complex mechanism involving multiple cationic LF molecules binding to a single anionic molecule of OPN. At least two classes of thermodynamically distinct LF binding sites were identified, with the higher affinity interactions (dissociation constants 10(-6)M) being in the biologically relevant range. Ca(2+) binding to OPN, or Fe(3+) binding to LF, influenced the enthalpy and entropy of binding, but had little effect on the overall binding affinity. Considering that the regions of electrostatic complementarity between OPN and LF mediate the numerous biological functions of each protein, we suggest that OPN may act as a carrier protein for LF in milk, and modulate the potent antimicrobial and immunostimulatory activities of the LF protein.