The nuclear magnetic resonance spectra of monoclonal Fab antibody fragments have been recorded in the absence and presence of the specific spin-label dinitrophenyl hapten. The difference spectra reveal the presence of about 50 amino acids in the region of the combining site. By selective deuteration and by use of double difference spectra, all the resonances in the spectral region -1 to 1.5 ppm have been identified. We have found that in the combining site region there are four or five valines, certainly three and possibly five threonines, three or four leucines, two or three isoleucines, and six or seven alanines. Selective deuteration of methionine and lysine reveals one methionine and two lysines in the difference spectra. All of these amino acids are estimated to be within 17 A of the paramagnetic hapten. By using difference spectra involving low fractional occupancy of the combining site with the spin-label hapten, it is established that one threonine and one valine are very close to the paramagnetic hapten.