NADPH-cytochrome c oxidoreductase and cyto- chrome P-450b mRNAs were enriched from tightly membrane-bound polysomes isolated from the livers of rats administered phenobarbital. This was accomplished by polysome immunoadsorption with monospe- cific I g G directed toward the oxidoreductase and cytochrome P-450b in conjunction with Staphylococcus aureus ghosts and oligo(dT)-cellulose chromatography. Double-stranded cDNA, synthesized from a preparation of poly(A)-RNA specifically enriched in both mRNAs, was inserted into the ampicillin resistance gene of pBR322 by use of oligo(dG)-oligo(dC) tailing. Clones containing DNA complementary to either oxidoreductase or cytochrome P-450b mRNA were se- lected by differential colony hybridization with [32P] cDNA synthesized from nonenriched, oxidoreductase mRNA-enriched, and cytochrome P-45Ob-mRNA-en-riched poly(A)-RNA. Plasmid DNAs isolated from clones which specifically annealed with either oxido- reductase-enriched cDNA or cytochrome P-450b cDNA were each verified by both a solution hybrid-arrest assay and a filter hybrid-selection assay. Restriction endonuclease maps were generated from oxidoreduc- tase and cytochrome P-450b cDNA plasmids that contained inserts of 1870 and 1830 base pairs, 4 to Methods used to isolate tightly membrane-bound polysomes and enrich specific polysomal mRNAs have been described in detail (13, 24,25) except for the following modifications. In order to enrich both oxidoreductase and cytochrome P-450b polysomal mRNAs, 5 mg each of purified anti-oxidoreductase IgG and anti-cytochrome P-450b IgG were added to 1500 AZW units of polysomes; after 3 h at 0 "C, Staphylococcus aureus ghosts (Calbiochem) were added and the adsorbed polysomes processed as described (24). Messenger RNA from this oxidoreductase and cytochrome P-450b dual mRNA-en-riched polysome preparation was isolated by oligo(dT)-cellulose chro- matography and subject to cDNA cloning into pBR322 using oligo(dG)-oligo(dC) tailing (24). In order to isolate separate prepara- tions of RNA enriched in oxidoreductase mRNA and cytochrome P450b mRNA, a sequential immunoadsorption scheme was utilized. First, oxidoreductase-synthesizing polysomes were removed from the phenobarbital-induced tightly membrane-bound polysome popula- tion by immunoadsorption of anti-oxidoreductase IgG-polysome complex. Immediately following sedimentation of the oxidoreductase po- lysome-S. aureus ghost complex, anti-cytochrome P-450 was added to the polysomes, and after a 3-h incubation at 0 "C, cytochrome P- 450b-synthesizing polysomes were immunoadsorbed and sedimented.