Alkylcatechol 2,3-dioxygenase was purified from the cell extract of recombinant Escherichia coli JM109 harboring the alkylcatechol 2,3-dioxygenase gene (bupB) cloned from the butylphenol-degrading bacterium Pseudomonas putida MT4. The purified enzyme (BupB) showed relative metacleavage activities for the following catechols: catechol (100%), 4-methylcatechol (572%), 4-n-butylcatechol (185%), 4-n-hexylcatechol (53%), 4-n-heptylcatechol (45%), 4-n-nonylcatechol (10%), 4-tert-butylcatechol (0%), and 3-methylcatechol (33%). The kinetic parameters, namely, K-m and V-max, for catechol, 4-methylcatechol, and 4-n-butylcatechol, were 23.4, 8.4, and 6.5 mu M and 25.8, 76.9, and 18.0 U mg(-1), respectively. These results suggest that BupB has broad substrate specificity for 4-n-alkylcatechols.