The N-terminal cellulose-binding domains CBD N 1 and CBD N 2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll β-sandwich structure with a cleft into which amorphous cellulose and soluble cellooligosaccharides bind. To determine the orientation of the sugar chain within these binding clefts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) spin-labeled derivatives of cellotriose and cellotetraose with isolated CBD N 1 and CBD N 2 was studied using heteronuclear 1 H- 1 5 N NMR spectroscopy. Quantitative binding measurements indicate that the TEMPO moiety does not significantly perturb the affinity of the cellooligo-saccharide derivatives for the CBDs. The paramagnetic enhancements of the amide 1 H N longitudinal (ΔR 1 ) and transverse (ΔR 2 ) relaxation rates were measured by comparing the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hydroxylamine (reduced) forms on the two CBDs. The bound spin-label affects most significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-cellotriose bound to CBD N 1 . This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBD N 1 and CBD N 2 in either orientation across their β-sheet binding clefts. The ratio of the association constants for binding in each of these two orientations is estimated to be within a factor of five to tenfold. This finding is consistent with the approximate symmetry of the hydrogen-bonding groups on both the cellooligosaccharides and the residues forming the binding clefts of the CenC CBDs.