We have used a plasmid containing DNA complementary to one of the two size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450 from rat liver to characterize and quantitate that mRNA. The plasmid used was constructed and identified as follows: Total poly(A+) RNA from 3-methylcholanthrene-induced liver was used as a template for cDNA synthesis. Double-stranded cDNA was inserted into plasmid pBR322 by the G-C tailing procedure. Recombinants were screened by colony hybridization using as probe [32P]cDNA prepared from partially purified cytochrome P-450 mRNA. A differential screening approach was used in which duplicate filters were hybridized with probe from either 3-methylcholanthrene treated or untreated rats. Plasmid p23 was strongly positive by colony hybridization. It was conclusively shown to contain cytochrome P-450 cDNA sequences by demonstrating that the mRNA which specifically hybridized to nitrocellulose-bound plasmid p23 could be translated in vitro into peptides that were immunoprecipitable with monoclonal antibodies specific for 3-methylcholanthrene-induced cytochrome P-450. The size and quantity of the mRNA complementary to plasmid p23 were determined by hybridization of the 32P-labeled plasmid to rat liver RNA that had been fractionated by electrophoresis under fully denaturing conditions and transferred to diazobenzyl-oxymethyl-paper. Plasmid p23 hybridized strictly to a single size of mRNA that was about 2000 nucleotides in length, the smaller of the two size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450. From this we concluded that, at least within the region of the mRNA contained within the insert of plasmid p23, the two size classes of 3-methylcholanthrene-induced cytochrome P-450 mRNA were very different in sequence. The mRNA complementary to plasmid p23 was increased about 4-fold after in vivo administration of 3-methylcholanthrene under conditions that result in maximal induction of 3-methylcholanthrene-induced cytochrome P-450 enzymatic activity. This increase in cytochrome P-450 mRNA parallels the increase in cytochrome P-450 enzymatic activity observed after 3-methylcholanthrene administration. These data suggest that the regulation of mRNA levels is an important point of control of cytochrome P-450 gene expression.