Analyses of the temperature dependences and shapes of nuclear magnetic relaxation dispersion (NMRD) signals of site-directed mutants of human myoglobin indicate that a water molecule is bound to the sixth coordination site of the ferric heme in proteins in which the valine at position 68 is changed to either aspartate (Val68Asp) or asparagine (Val68Asn). Both NMRD data and electron paramagetic resonance (EPR) spectra show that carboxylate is axially ligated to Fe(III) in the Val68Glu mutant. The EPR spectra of the Val68Asp and Val68Asn derivatives show much smaller rhombic splittings than the spectrum of the Val68Glu protein.