The relative rates of transcription of epoxide hydratase, NADPH-cytochrome P-450 oxidoreductase, and cytochrome P-450b genes were determined in purified hepatocyte nuclei isolated at different times after phenobarbital administration. The rates of transcription of the epoxide hydratase and oxidoreductase genes were increased 4- and 9-fold, respectively, above control levels 1 h after administration of the drug. Transcription rates for the oxidoreductase gene slowly decreased to control values at 24 h while transcription of the epoxide hydratase gene rapidly declined to approximately 2-fold above control values at 2.5 h and remained at this level up to 24 h after administration. Interestingly, transcription of the cytochrome P-450b gene followed a markedly different induction time course than the epoxide hydratase and oxidoreductase genes. Specifically, cytochrome P-450b gene transcription increased 23-fold above control values 6 to 8 h after phenobarbital administration and then slowly declined to 14-fold at 24 h. The increased levels of intranuclear pre-mRNA and cytoplasmic mRNA for each enzyme correlated well with transcriptional activity. In contrast to these results, the rate of transcription of the serum albumin gene was not affected, and levels of albumin mRNA actually decreased after administration of phenobarbital. The rapid increase in the rates of transcription and the appearance of elevated levels of nuclear pre-mRNAs and cytoplasmic mRNAs unequivocally demonstrates that phenobarbital elevates levels of these drug-metabolizing enzymes primarily by augmenting the rate of transcription of their respective genes.