Remarkably high levels of cytochrome P450 aromatase (P450arom) enzyme are expressed in the brains of teleost fish when compared to the ovaries of the same fish, or to the brain or ovaries of other vertebrates. Northern analysis using a full-length P450arom cDNA from a goldfish brain library indicates high accumulated levels of CYP19 mRNA in the brain but fails to detect P450arom mRNA in the ovary. The possibility of different brain and ovarian mRNA variants was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) amplification of ovarian RNA using degenerate primers led to the isolation of a 243 bp P450arom cDNA fragment with approximately 20% of nucleotide and amino acid replacements when compared to the brain cDNA. Southern analysis with sequence-specific probes indicated two distinct CYP19 loci, and this was confirmed by PCR-restriction enzyme analysis of genomic DNA. Corresponding brain- and ovary-type genomic sequences were identified in a second, diploid fish species (zebrafish), evidence that two genes are not caused per se by tetraploidy in goldfish. RT-PCR analysis of different tissues with sequence-specific primers showed high levels of the brain mRNA variant and much lower levels of the ovarian variant in neural tissues with high enzyme activity. In contrast, the ovary expressed low levels of the ovarian mRNA variant exclusively. The data imply that the expression of two CYP19 genes in goldfish is controlled by distinct regulatory mechanisms. Further studies are required to determine whether the two genes lead to functionally distinct isozymes.