Free energy simulations and standard molecular mechanics calculations have been used to examine the binding specificity of the C-type lectin rat mannose-binding protein. This protein, which is important in inflammation and immunoglobin-independent immune response, recognizes and binds to mannose and high-mannose oligosaccharides but not to galactose. Molecular dynamics simulations were used to estimate the free energy difference between these two sugars in aqueous solution and in the binding site of the protein. While the simulations found mannose to be favored over galactose, interactions with the solvent tended to favor galactose : The calculated binding free energy difference for these two sugars to MBP was 3.1 kcal/mol favoring mannose, approximately twice the experimental value. An estimate of the resolution into components found that enthalpy favored mannose but that entropy favored galactose, a reversal of the situation in solution. From standard MD simulations, the galactose was found to rotate in the binding site away from the mannose orientation found in crystallographic studies.