The transforming growth factor (TGF)¹ family of secretory polypeptides comprises signaling proteins involved in numerous physiological processes including vascular development and vessel wall integrity. Both pro- and anti-angiogenic effects of TGF-1 have also been documented. To study the intracellular mechanisms involved in capillary tube morphogenesis, endothelial cell aggregates were cultured in a fibrin matrix. It was found that the pattern of capillary tubes formed in a fibrin matrix was altered in response to TGF-1 treatment such that the capillary-like structures displayed a bipolarized pattern. In contrast, in untreated control and fibroblast growth factor 2-treated cells the pattern of capillary tubes formed was random. TGF-1 also downregulated urokinase type plasminogen activator (uPA) activity while upregulating plasminogen activator inhibitor I (PAI-1) and thrombospondin 1 (TSP1) gene expression. To investigate the signaling cascade mediating the phenotypic changes observed, pharmacological inhibitors of p38 MAPK, Sp1 transcription factor, and c-Jun N-terminal kinase (JNK), and the cytokine tumor necrosis factor (TNF-) were used. The p38 MAPK inhibitor SB203580 reversed the TGF-1-dependent inhibition of uPA activity but not its morphogenetic effect. In contrast, the DNA intercalator WP631 and TNF- counteracted the TGF-1-induced morphogenetic effect while the JNK inhibitor SP600125 effectively inhibited capillary tube formation. These results indicate that the TGF-1-induced capillary tube pattern is independent of the p38 MAPK-activated PAI-1 and TSP1 expression, but the mechanism involves Sp1-dependent transcriptional regulation. The results also raise the possibility that the JNK pathway, which controls convergent extension in Xenopus, may be involved in vessel wall patterning in mammalian systems.