Amino acid residues in the active site of quinoline 2-oxidoreductase (Qor) that are deemed important for substrate binding and turnover were replaced by site-directed mutagenesis. The apparent k(cat) values for quinoline were reduced 2.4-, 38, 40-, and 199-fold in the protein variants QorA259G, QorW331G, QorV373A, and QorA546G, respectively. The substitution A259G did not significantly alter K-m (app.) Despite the presumed crucial role of W331 and V373 in substrate positioning, the replacements W331G (K-m (app): 0.33 mm) and V373A (K-m (app) : 0.41 mm) only slightly affected affinity for quinoline (K-m (ap), of Qor: 0.12 mm). QorA546G showed an increased affinity for quinoline and quinoxaline, as suggested by its 4.3- and 7.5-fold decrease in K-m (app) (quinoline) and K-m (app) (quinoxaline), respectively, compared with Qor. The relative activities of the protein variants towards substituted quinolines differed from those of Qor. QorW331G, for example, may be suitable for hydroxylation of quinoxaline and C4-substituted quinolines.