Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and eNOS from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). We investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 hr after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 hr after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, SNAREs and SNAPs which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain; syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23 and ~{&A~}-SNAP) in the enlarged/circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, N-ethylmaleimide sensitive factor (NSF), an ATPase required for the disassembly of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus, MCT-induced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs and SNAPs and of intracellular vesicular trafficking.