Endothelium-dependent relaxation in rabbit aorta was inhibited by exposure to two cytochrome P-450 inhibitors, metyrapone and SKF-525A. Aortic rings were contracted to a stable plateau by addition of phenylephrine (10<sup>–7</sup> M). Relaxation was elicited by the cumulative addition of methacholine (3 × 10<sup>-8</sup>–3 × 10<sup>–6</sup> M) or A23187 (10<sup>–8_</sup>10<sup>–6</sup> M). Exposure to metyrapone (500 µM) or SKF-525A (10 µg/ml) was found to inhibit relaxation in response to concentrations of methacholine exceeding 10<sup>–7</sup> M. Maximal relaxation was inhibited 73% by metyrapone. Relaxation stimulated by concentrations of A23187 exceeding 10<sup>–7</sup> M was also found to be inhibited by both metyrapone and SKF-525A exposure. Maximum A23187-induced relaxation (55% of the phenylephrine contractile response) was inhibited 40% by metyrapone and 55% by SKF-525A. Arachidonic acid (10–100 µ M) also elicited endothelium-dependent relaxation in rings pretreated with indomethacin (10 µg/ml) and contracted with phenylephrine. This relaxation response was abolished by exposure to metyrapone or SKF-525A. These results suggest that cytochrome P-450 may be involved in endothelium-dependent relaxation responses, perhaps by metabolizing arachidonic acid to active products.