The binding of Cu(II) to a lipophilic bis-hydroxamate binder, RL252, and its parent RL239, was investigated by pulsed EPR. The binders have two arms, each terminated with a hydroxamate group which serves as a donor. The major difference between RL252 and RL239 is the absence of the leucine amino acid bridge in RL239. Orientation-selective electron spin echo envelope modulation (ESEEM) experiments were carried out at 8.45 and 9.15 GHz. The spectra obtained were exceptionally well resolved and indicated that the cancellation condition, which requires that the hyperfine coupling is approximately twice the nuclear Larmor frequency, is met at 8-9 GHz. The spectra of both Cu-RL252 and Cu-RL239 showed the nuclear quadrupole resonance (NQR) frequencies, nu(0), nu-, and nu(+), of the N-14 of the hydroxamate groups at 1.6-1.7, 2.15-2.25, and 3.8-3.9 MHz. A peak corresponding to the double quantum transition in the "noncanceled manifold", nu(DQ), was observed at 5-5.2 MHz, and at some magnetic fields a peak corresponding to a single quantum transition, nu(SQ), and its combination harmonic appeared as well. The assignment of all the ESEEM frequencies was achieved by the application of the two-dimensional hyperfine sublevel correlation (HYSCORE) experiment. Following the assignment, simulations of the orientation-selective ESEEM spectra were performed, yielding the magnitude and orientation of the quadrupole and hyperfine tensors of the coupled nitrogens. While only one best fit set of quadrupolar parameters was found, two such sets were obtained for the hyperfine interaction. Analysis of the orientation of the quadrupole tensor with respect to the g-tensor showed that in both Cu-RL252 and Cu-RL239 the binding site is close to coplanar. Very subtle differences in the spin Hamiltonian parameters were observed between Cu-RL252 and Cu-RL239, The N-14 quadrupolar parameters and the anisotropic hyperfine component were slightly larger in Cu-RL239. The relatively small N-14 hyperfine coupling is attributed to a node in the molecular orbital, occupied by the unpaired electron, at the nitrogen.