The ω-hydroxylation product of arachidonic acid is thought to be a potent vasoconstrictor or a precursor thereof in kidney. In this report, we have measured the capacity of four rabbit CYP4A enzymes, each expressed in COS-1 cells, to catalyze the ω-hydroxylation of arachidonic acid. These rates were compared to those obtained for other substrates such as lauric acid, palmitic acid, and prostaglandins PGE1 and PGA1. With the exception of P4504A5, all of the enzymes tested exhibited relatively high rates for the ω-hydroxylation of arachidonic acid. P4504A5 showed very little activity toward arachidonic or palmitic acids as compared to that toward lauric acid (<10%). In contrast, P4504A6 and P4504A7 catalyzed the ω-hydroxylation of arachidonic acid at rates that were roughly 50% of that observed for lauric acid. P4504A4 was not active toward lauric acid, but it also catalyzed the ω-hydroxylation of arachidonic acid at a rate that was roughly 20% of that exhibited for PGE1. Thus, each enzyme exhibits a distinct substrate specificity profile across this panel of substrates. A sensitive RNase protection assay was used to provide a more quantitative estimate of the relative abundance of mRNAs encoding P4504A5, P4504A6, and P4504A7 in liver and kidney from control, pregnant, and clofibrate-treated animals. CYP4A5 is the most abundant of the mRNAs, but it was not induced in kidney and only moderately (2-fold) in liver by clofibric acid. CYP4A7 exhibits a similar pattern of induction by clofibrate. In contrast, CYP4A6 is induced 12-fold in liver and 6-fold in kidney. The higher induction ratio largely reflects a lower basal level of expression for CYP4A6 than for CYP4A7 and CYP4A5. Following treatment with clofibrate, the amount of CYP4A6 mRNA is similar to those of CYP4A5 and CYP4A7. Pregnancy did not affect the expression of CYP4A5, CYP4A6, or CYP4A7, although it induced the expression of CYP4A4 to detectable levels in the liver and kidney, where it is not normally found in nonpregnant animals. Our results indicate that the enzyme whose mRNA is most highly induced by clofibric acid (P4504A6) and the enzyme selectively elevated during pregnancy (P4504A4) both exhibit relatively high rates for the ω-hydroxylation of arachidonic acid.