The content and spectral characteristics of cytochrome P450 (Cyt P450) and cytochrome b 5 (Cyt b 5) extracted from shoots of etiolated maize and rice seedlings were studied by using ultraviolet (UV) difference spectrophotometry. The results showed that fenclorim, rac-metolachlor and S-metolachlor may induce the same P450 isoenzyme with λmax at 453 nm, while naphthalic anhydride (NA) induced another one with λmax at 447 nm. The microsomal Cyt P450 and Cyt b 5 content of maize seedlings was higher than that of rice, and the Cyt b 5 content was higher than that of Cyt P450. Maize and rice microsomal Cyt P450 and Cyt b 5 were induced at different levels by the four chemicals, with the order as follows: NA > fenclorim > rac-metolachlor > S-metolachlor with p < 0.05. When induced by NA, fenclorim, rac-metolachlor and S-metolachlor, the maize Cyt P450 content was, respectively, 5.63-, 3.30-, 3.02- and 2.48-fold that of the control, the rice Cyt P450 content was 8.54-, 2.20-, 1.91- and 1.33-fold that of the control, the maize Cyt b 5 content was 9.89-, 5.49-, 4.69- and 3.40-fold that of the control, and the rice Cyt b 5 content was 7.76-, 4.56-, 2.60- and 1.82-fold that of the control. An enantio-difference existed when rac- and S-metolachlor combined with plant Cyt P450. The interaction of microsomal Cyt P450 with S-metolachlor is higher than that with rac-metolachlor, which may be one of the reasons why S-metolachlor is superior at killing weeds compared with rac-metolachlor. These results will help to develop an understanding of the tolerance for and selectivity of rac- and S-metolachlor.