Transformed endothelial cells expressing the polyoma middle T oncogene (endothelioma cells) cause tumors in mice and recruit host endothelial cells without stimulating their proliferation. A role for the endothelial vasoactive mediators nitric oxide (NO) and endothelin-1 (ET-1) in tumor cell biology has been postulated. Thus, the existence of a synthesizing pathway for both mediators in endothelioma cell lines derived from both subcutaneous (sEnd.1) and thymic (tEnd.1) hemangiomas was investigated. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in both tumor cell lines. Moreover, ET-1 immunoreactive material was found in culture supernatants and reverse phase HPLC of the conditioned media revealed two peaks corresponding to the retention times of synthetic ET-1 and big ET-1. Endothelioma cell lines strongly inhibited thrombin-induced platelet aggregation. The antiplatelet activity was cell-number dependent, and could be potentiated when endothelioma cells were stimulated with either carbachol (100 μM) or histamine (100 μM). In addition, both agonists evoked a transient rise in the intracellular [Ca2+] of these cells. Incubation of endothelioma cells in L-arginine deficient medium (24 h) or pretreatment with the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100μM) significantly reduced their antiplatelet activity. The inhibitory effect of L-NAME was overcome by co-incubation with L- (100 μM) but not D-arginine (100 μM). Conversely, exposure of endothelioma cells to either the protein synthesis inhibitor cycloheximide (1 μM, 48 h) or the glucocorticoid dexamethasone (1 μM, 48 h) did not modify their antiplatelet activity. The presence of constitutive, endothelial NO-synthase was confirmed by western blot of cell extracts from both cell lines. These results demonstrate that both ET-1 and NO are synthesized by and released from transformed endothelial cells. They further suggest that the enzyme responsible for NO synthesis in endothelioma cells is constitutively expressed. © 1994, Informa UK Ltd. All rights reserved.