Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by HPLC and used for binding studies with ATP and pyridoxal analogues. The spectroscopic properties of trinitrophenyl-ATP bound to the 24-kDa fragment are indistinguishable from those of TNP-ATP bound to the native kinase. The small 16-kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the analogues. The same pattern of digestion was observed when IAF pyridoxal kinase, carrying a fluorescent probe covalently bound to a specific SH residue, was preincubated with chymotrypsin. The kinetics of proteolysis of IAF-pyridoxal kinase was monitored by emission anisotropy, and the analysis of the initial rate of proteolysis at various concentrations of chymotrypsin reveals that the rate of unfolding of native pyridoxal kinase plays a dominant role in the proteolytic process.