Orientation selective and multiple frequency ESEEM experiments on substrate free cytochrome P450cam (CP450cam) with O-17-enriched water are reported. The O-17 ESEEM frequencies were obtained from Fourier transformation of the ratio of ESEEM waveforms of CP450cam with enriched water and CP450cam with nonenriched water. Numerical simulations were carried out to determine the isotropic and the anisotropic hyperfine interactions and the quadrupole interaction of the O-17. From the magnitude (e(2)-qQ/h = 6.6 MHz) and asymmetry (eta = 0.95) of the O-17 quadrupole interaction, we conclude that the distal axial ligand of Fe3+ in the CP450 heme is a water molecule. Moreover, from the orientation of the O-17 quadrupole tenser, the orientation of the water molecule was found to be confined (within +/-10 degrees) with respect to the Fe-N directions within the heme plane. Two possible sets of isotropic and anisotropic components of the O-17 hyperfine interaction, (+/-2.6, +/-0.3 MHz) and (+/-0.4, +/-1.8 MHz), were found to satisfactorily reproduce the experimental results. Both sets indicate very small O-17 hyperfine couplings which is consistent with the unpaired electron residing predominately in the d(yz) orbital of the Fe3+. Ligand field models for each set are presented.