Upon titration with palmitate, the (1)H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb) with fatty acid. Other detectable hyperfine shifted resonances of the heme group remain unchanged. Mb also enhances fatty acid solubility, as reflected in a more intense methylene peak of palmitate in Mb solution than in Tris buffer. Ligand binding analysis indicates an apparent palmitate dissociation constant (K(d)) of 43microM. These results suggest that Mb can bind fatty acid and may have a role in facilitating fatty acid transport in the cell.