Pulmonary arteries from the Madison (M) strain relax more in response to acetylcholine (ACh) than those from the Hilltop (H) strain of Sprague-Dawley rats. We hypothesized that differences in endothelial nitric oxide (NO) synthase (eNOS) expression and function, metabolism of ACh by cholinesterases, release of prostacyclin, or endothelium-derived hyperpolarizing factor(s) (EDHF) from the endothelium would explain the differences in the relaxation response to ACh in isolated pulmonary arteries. eNOS mRNA and protein levels as well as the NO-dependent relaxation responses to thapsigargin in phenylephrine (10⁶ M)-precontracted pulmonary arteries from the M and H strains were identical. The greater relaxation response to ACh in M compared with H rats was also observed with carbachol, a cholinesterase-resistant analog of ACh, a response that was not modified by pretreatment with meclofenamate (10⁵ M). N-nitro-L-arginine (10⁴ M) completely abolished carbachol-induced relaxation in H rat pulmonary arteries but not in M rat pulmonary arteries. Precontraction with KCl (20 mM) blunted the relaxation response to carbachol in M rat pulmonary arteries and eliminated differences between the M and H rat pulmonary arteries. NO-independent relaxation present in the M rat pulmonary arteries was significantly reduced by 17-octadecynoic acid (2 µM) and was completely abolished by charybdotoxin plus apamin (100 nM each). These findings suggest that EDHF, but not NO, contributes to the strain-related differences in pulmonary artery reactivity. Also, EDHF may be a metabolite of cytochrome P-450 that activates Ca²⁺-dependent K⁺ channels.