Camel lens zeta-crystallin is an NADPH:quinone oxidoreductase, showing limited quinone substrate specificity: among the quinones tested, the orthoquinones were the best substrates. The kinetic mechanism of NADPH:quinone oxidoreductase activity of zeta-crystallin was investigated by steady-state initial velocity and product-inhibition studies. The results were consistent with the enzyme having a ping-pong mechanism and the intrinsic K(m) values for NADPH and 9,10-phenanthrenequinone (PQ) were estimated to be 48.0 +/- 2.5 and 36.0 +/- 1.5 microM, respectively. NADP+ showed a mixed type of inhibition with respect to NADPH, while it was a competitive inhibitor with respect to PQ. Dithiothreitol (DTT) was a strong competitive inhibitor with respect to PQ with a Ki value of 50 microM. Similarly, 2, 3-dimercaptopropanol inhibited the enzyme activity with a Ki value of 260 microM. The enzyme was inactivated by sulfhydryl groups, modifying agents such as 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) and N-ethyl-maleimide. The substrate specificities and kinetic properties of camel lens NADPH:quinone oxidoreductase reported here distinguish it from previously characterized quinone oxidoreductases. This paper reports, for the first time, on the kinetic mechanism of camel lens zeta-crystallin.