Preparations of mRNA isolated from rabbit lung and liver were used in the construction of libraries that were screened for cDNAs encoding the pulmonary or hepatic isozyme of the flavin-containing monooxygenase. The hepatic library was screened with cDNA encoding the flavin-containing monooxygenase expressed in pig liver, and a clone containing a 2.0-kilobase insert was detected and isolated. This cDNA insert encoded a protein of 535 amino acids with a primary structure 87% identical to that of the pig flavin-containing monooxygenase. The pulmonary library was screened with polyclonal antibodies to the flavin-containing monooxygenase expressed in rabbit lung, and a clone containing a 2.6-kilobase insert was detected and isolated. Although the protein encoded by this insert also contained 535 amino acids, its primary sequence was only 56% identical to that of the liver enzyme. The sequences of several peptides obtained by digestion of the purified rabbit pulmonary flavin-containing monooxygenase with trypsin matched exactly with sequences derived from the cDNA structure. Tissue-specific distribution of mRNA for the hepatic and pulmonary isozymes of the flavin-containing monooxygenase was consistent with the distribution of protein, an indication that expression of flavin-containing monooxygenase is controlled at the level of transcription. Analysis of genomic DNA indicates that both the hepatic and pulmonary enzymes may be products of single genes.