The regulation of prostacyclin (PGl₂) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGl₂ was monitored by RIA of 6‐keto PGF_1α and dose‐dependent increases observed with human α‐and γ‐thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6‐keto PGF1α approximating responses with 1 μM γ‐thrombin, 5μM arachidonate, or 10 μM histamine. Diisopropyl phosphorofluo‐ridate‐inactivated α‐thrombin did not stimulate PGl₂ release, demonstrating that catalytic activity was required for thrombin‐stimulated PGl₂ release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGl₂ synthesis in a dose‐dependent and time‐dependent manner (20 mM NaF, 4.4 ± 0.5‐fold increase at 10 min, 11.9 ± 1.5‐fold increase at 30 min). Neither α‐thrombin nor NaF‐stimulated PGl₂ release was dependent upon the availability of extracellular Ca^{+ +}. The hypothesis that G proteins are involved in agonist‐stimulated PGl₂ synthesis was further supported by studies using digitonin‐permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5′‐0‐3‐thiotrisphosphate (GTP γ S), which effected significant dose‐dependent increases in PGl₂ synthesis compared with control levels of 6‐keto PGFα. In contrast, the G‐protein inhibitor GDP β S, (guanosine 5′‐0‐2‐thiodiphosphate), attenuated α‐thrombin‐mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 μg/ml) did not inhibit the PGl₂ synthesis stimulated by either α‐thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross‐reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the α‐subunit of other G‐proteins. Preincubation of HUVEC microsomal membranes with α‐thrombin diminished pertussis toxin‐catalyzed ADP ribosylation in a time‐dependent manner. These data suggest that thrombin stimulation of PGl₂ synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin‐occupied receptor is coupled to phospholipase activities by a pertussis toxin‐insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.