The conversion of androgens to estrogens is catalyzed by a complex of enzymes that includes a specific cytochrome P-450 aromatase (P-450arom). In this paper we describe the high level expression of aromatase activity in the rat Leydig cell tumor line, R2C. We also report the isolation of cDNA clones encoding the rat aromatase P-450arom from a cDNA library prepared from this cell line. Analysis of these cDNA clones predicts a protein sequence with a high degree of sequence conservation when compared to the chicken and human P-450arom enzymes. Notably, four of the cDNA clones were found to lack the last coding exon that contains the heme-binding domain, a structural feature essential for aromatase activity. These clones were found to contain instead a segment of genomic DNA derived from an unspliced intron. Northern analysis using a fragment of the coding region of the rat P-450arom cDNA as probe revealed that three species of P-450arom mRNa are expressed in rat ovary that are similar to those identified in RNA samples prepared from the rat R2C cell line. Analysis of the same samples of RNA using a probe derived from the 3' terminal intron segment of the rat aromatase cDNA clones that lack the heme-binding domain indicates that two of the species of aromatase mRNA transcripts present in both rat ovary and R2C cell lack the heme-binding domain and thus must encode a nonfunctional aromatase protein. These findings have important implications for the measurement of aromatase mRNA and appear to explain why three sizes of rat P-450arom mRNA exist on Northern analysis and why previous studies failed to demonstrate a clear relationship between aromatase mRNA, protein, and enzymatic activity in the rat ovary.