Cultures of established cell lines, derived from rat and human hepatomas, were exposed to dibenzofuran (DBF) or monochlorodibenzofuran isomers (Cl-DBFs) and the induction of cytochrome P-450-dependent monooxygenases was examined. When rat (H4-II-E-C3) cell cultures were incubated with 2-Cl- or 3-Cl-DBF, the activities of arylhydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase increased progressively and after 20 hr of incubation reached a maximum, while benzphetamine N-demethylase activity was scarcely induced. The maximum AHH activities were 15.1 and 7.9 times the initial values, after incubation with 3-Cl- and 2-Cl-DBFs, respectively. DBF also increased AHH activity, to a maximum, 2.7 times the initial activity, after 14 hr of incubation. Similar results were obtained when human (Chang liver) cell cultures were incubated with DBF, 2-Cl- or 3-Cl-DBF. To determine relative induction potencies, rat cell cultures were incubated with 25-150 μm of 1-Cl-, 2-Cl-, 3-Cl- or 4-Cl-DBF for 20 hr. Both the AHH and 7-ethoxycoumarin O-deethylase activities were increased in a dose-dependent manner by 2-Cl-, 3-Cl- and 4-Cl-DBF (in the order 3-Cl- > 2-Cl- = 4-Cl-). The immunoreactive cytochrome P-450c of rat cells was similarly increased by incubation with 2-Cl-, 3-Cl or 4-Cl-DBF (3-Cl- > 2-Cl- = 4-Cl-). 1-Cl-DBF increased neither AHH activity nor cytochrome P-450c. These results indicate that isomers of Cl-DBFs induce effectively 3-methylcholanthrene-inducible type of cytochrome P-450.