High-resolution proton-detected heteronuclear correlation NMR spectroscopy allows the measurement of N-15 spin relaxation rates at multiple sites throughout a biological macromolecule. The rate constants are determined by stochastic internal motions on time scales of picoseconds to nanoseconds, overall molecular rotational diffusion on time scales of nanoseconds, and chemical exchange rates on time scales of microseconds to milliseconds. A new method has been developed for distinguishing the contributions of chemical exchange from the contributions due to anisotropic rotational diffusion by measuring both longitudinal and transverse interference between the H-1-N-15 dipolar and N-15 chemical shift anisotropy interactions. The spectroscopic experiment for measuring the longitudinal cross-correlation rate constant for H-1-N-15 dipolar/N-15 chemical shift anisotropy interference is based on the approach for measuring the transverse cross-correlation rate constant (Tjandra, N,; Szabo, A.; Bar, A. J. Am. Chem. Sec. 1996, 118, 6986-6991)but incorporates a novel method for averaging the relaxation rates of longitudinal magnetization and two spin order. Application of this technique to Escherichia coli ribonuclease H affords an improved description of rotational diffusion anisotropy and permits a more accurate assessment of chemical exchange in this molecule. The results definitively demonstrate that amino acid residues K60 and W90 are subject to conformational exchange processes, whereas increased transverse relaxation rates for residues in the helix alpha(D) arise from anisotropic rotational diffusion.