20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt⁻¹ ⋅ day⁻¹iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.