W-band (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) spectroscopic techniques were used to determine the hyperfine couplings of different protons of Cu(II)histidine complexes in frozen solutions. The results were then used to obtain the coordination mode of the tridentate histidine molecule and to serve as a reference for Cu(II)-histidine complexation in other, more complex systems. Cu(LI) complexes with L-histidine and DL-histidine-alpha -d,beta -d(2) were prepared in H2O and in D2O and orientation-selective W-band H-1 and H-2 pulsed ENDOR spectra of these complexes were recorded at 4.5 K. These measurements lead ro the unambiguous assignment of the signals of the H-alpha, H-beta, imidazole H-epsilon, and the exchangeable amino, H-am, protons. The N-14 superhyperfine splitting observed in the X-band EPR spectrum and the presence of only one type of H-alpha and H-beta protons in the W-band ENDOR spectra show that the complex is a symmetric bis complex. Its g(parallel to) is along the molecular symmetry axis, perpendicular to the equatorial plane that consists of four coordinated nitrogens in histamine-like coordinations (NNNN). Simulations of orientation-selective ENDOR spectra provided the principal components of the protons' hyperfine interaction and the orientation of their principal axes with respect to gu. From the anisotropic part of the hyperfine interaction of H-alpha and H-beta and applying the point-dipole approximation, a structural model was derived. An unexpectedly large isotropic hyperfine coupling, 10.9 MHz, was found for H-alpha. In contrast, H-alpha of the Cu(II)-1-methyl-histidine complex, where only the amino nitrogen is coordinated, showed a much smaller coupling. Thus, the hyperfine coupling of H-alpha can serve as a signature for a histamine coordination where both the amino and imino nitrogens of the same molecule bind to the Cu(IT), forming a six-membered chelating ring. Unlike H-alpha the hyperfine coupling of H-epsilon is not as sensitive to the presence of a coordinated amino nitrogen of the same histidine molecule.