The cDNAs encoding cytochromes CYP 4A2 and 4A3 were cloned by RT-PCR amplification of male rat kidney and liver RNAs, respectively. Sequence analysis demonstrated that these cDNAs were nearly identical to the published sequences for CYPs 4A2 and 4A3. CYP 4A2 and 4A3 share extensive sequence homology that extends into their 3'- and 5'-untranslated segments ( approximately 97% overall nucleotide identity). Analysis of cDNA and genomic DNA sequences shows that a sequence of 123 bp, recognized as an intron during the processing of CYP 4A2 transcripts, is conserved in the 4A3 mRNAs and that these otherwise highly homologous genes show different exon-intron distributions. The CYP 4A2 and 4A3 cDNAs were expressed in a baculovirus-insect cell expression system. Purified recombinant CYP 4A2 oxidized arachidonic acid to a mixture of 19- and 20-hydroxyeicosatetraenoic acids (20 and 80% of the total products, respectively). Reaction rates were maximal when CYP 4A2 was reconstituted in the presence of an equimolar concentration of cytochrome b5 and a 10-fold molar excess of NADPH-cytochrome P450 reductase. Studies using microsomal fractions isolated from noninfected insect cells and from cells infected with CYP 4A3 recombinant baculoviruses showed (a) the presence of an endogenous lauric acid omega-hydroxylase and arachidonic acid epoxygenase in the noninfected cells, (b) the CYP 4A3-dependent oxidation of lauric acid to 11- and 12-hydroxylaurate (24 and 76% of the total products, respectively), and (c) the lack of arachidonic acid metabolism by microsomal recombinant CYP 4A3. Nucleic acid hybridization and immunoelectrophoresis studies demonstrated that (a) CYP 4A2 transcripts are abundantly expressed in the female kidney and that CYP 4A3 is expressed in female but not in male liver, (b) anti-CYP 4A2 immunoreactive material was detected only in the male kidney, (c) male and female livers or kidneys support only low levels of CYP 4A3 translation, and (d) excess dietary salt does not alter the kidney levels of mRNA transcripts encoding CYP 4A1, 4A2, or 4A3 or change the levels of microsomal anti-4A1 or -4A2 immunoreactive proteins. Finally, no significant differences were observed between Dahl salt resistant or Dahl salt sensitive rats in the levels and/or salt regulation of mRNA transcripts enecoding CYP 4A1, 4A2, or 4A3 or the in levels of the corresponding proteins.