1. Pertussis toxin inactivates Gi-protein, which mediates the inhibitory effects of receptors on adenylate cyclase. The effects of the toxin on endothelium-dependent and independent relaxations were determined in porcine coronary arteries. 2. Arterial rings (with and without endothelium) were suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution (maintained at 37 degrees C, gassed with 95% O2 and 5% CO2). 3. Incubation of the tissues with pertussis toxin (100 ng/ml for 60 min) virtually abolished the endothelium-dependent relaxations produced by the alpha 2-adrenergic agonist, UK 14304, and by 5-hydroxytryptamine. Endothelium-dependent relaxations to thrombin and to aggregating platelets were markedly reduced, whereas those produced by bradykinin were only minimally affected. Endothelium-dependent responses produced by the calcium ionophore (A23187) and by adenosine diphosphate were not altered by pertussis toxin. 4. Pertussis toxin did not affect the direct, endothelium-independent relaxations produced by nitric oxide, or by adenosine diphosphate. 5. These experiments demonstrate that pertussis toxin interferes with the release of endothelium-derived relaxing factor(s) evoked by certain, but not all, endothelial activators. The release of endothelium-derived relaxing factor(s) may occur through different pathways involving Gi-protein-dependent and independent mechanisms.