The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations. The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions. In addition, the position of oxidation and the enantiomeric composition of products were characterized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity. Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes. A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I). The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO.