The purpose of this study is to determine which pertussis toxin-sensitive guanine nucleotide-binding protein (Gi) mediates alpha 2-adrenergic receptor stimulation of endothelium-derived relaxing factor (EDRF) release. Bovine aortic endothelial cells were treated with pertussis toxin (0-100 ng/ml) for 16 h and stimulated with an alpha 2-adrenergic receptor agonist, UK14304, to release EDRF in a bioassay system. Pertussis toxin produced a concentration-dependent decrease in EDRF release with maximal inhibition (80%) occurring at 5 ng/ml. This correlated with a decrease in receptor-G protein coupling as measured by 87% loss of high affinity agonist binding sites and 94% decrease in agonist-stimulated GTPase activity. Immunoprecipitation of [32P]NAD-ribosylated membranes using specific Gi protein antisera demonstrated that complete ADP-ribosylation of Gi alpha 2 occurred at 5 ng/ml compared to 30 ng/ml for Gi alpha 3. When bovine aortic endothelial cell membranes were treated with carboxyl terminus-directed antisera to Gi alpha 2 (P4) and Gi alpha 3 (EC), the P4 antisera abolished 86% of the high affinity agonist binding sites and 93% decrease in agonist-stimulated GTPase activity, while the EC antisera had minimal effect (12%). These results indicate that Gi alpha 2 mediates most of the EDRF released via the alpha 2-adrenergic receptor.