An antibody elicited against the trans, syn uracil cyclobutane dimer hapten 1 catalyzes the light-dependent cleavage of uracil dimers 1 and 2 to the corresponding monomers 3 and 4. Kinetic analysis of the antibody-catalyzed reaction affords a value of k(cat)/K-M = 1.7 x 10(3) M(-1) min(-1) for substrate 2, and comparison to the uncatalyzed reaction gives a rate acceleration of k(cat)/k(uncat) = 380. The wavelength dependence of the reaction and fluorescence quenching behavior suggest that a tryptophan residue is acting as a photosensitizer. The reaction mechanism was probed by measurement of secondary deuterium isotope effects. Substrates with selective deuterium substitutions in the cyclobutane ring were prepared, and isotope effects were measured by the method of internal competition using electrospray-ionization mass spectrometry to quantify the products. Kinetic isotope effects of (alpha-D)(V/K) = 1.11, 1.14, and 1.20 were observed for the 5,5’-, 6,6’-, and 5,5’,6,6’-labeled substrates, respectively. These results are comparable to those observed in a similar study on the E. coli enzyme DNA photolyase(1) and suggest that the reaction may proceed via a radical anion intermediate with concerted breakage of the 5,5’ and 6,6’ bonds, Alternatively the reaction may proceed via a mechanism in which the first bond is cleaved in a reversible fashion.