The sensitivity of the 3-methylcholanthrene (MC)-mediated induction of aryl hydrocarbon hydroxylase (AHH) activity to pretreatment with inhibitors of protein and RNA synthesis was studied. Cordycepin significantly inhibited the induction at 12 h but not at 3 h post-treatment with MC. Both cycloheximide and actinomycin D significantly inhibited the MC-mediated induction at 3 and 12 h after MC treatment. An MC-mediated stimulation of 3H-leucine incorporation into microsomal protein in vivo was observed at 12 h following MC treatment. A cell-free protein synthesis system employing intestinal ribosomes was developed. The rate of protein synthesis (3H-leucine incorporation/mg rRNA/3 min) and the number of active ribosomes (3H-peptidyl-puromycin formed/mg rRNA) were determined. No effect of in vivo MC treatment was observed in subsequently isolated intestinal ribosomes at either 3, 9 or 18 h post-treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on intestinal microsomes prepared from control, corn-oil- and MC-treated rats 18 h after treatment. The microsomal proteins depicted only slight quantitative changes in microsomal proteins following MC treatment, and no unique protein peaks. These results may explain the lack of detectable stimulation of the activity of isolated ribosomes. In addition, the methods used may lack sufficient sensitivity to detect the small net change in protein necessary to account for the increase in AHH activity seen after MC induction.