Adipose triglyceride lipase (ATGL) was identified in 2004 as a key enzyme in triglyceride metabolism. Systemic deletion of the ATGL gene in mice leads to massive accumulation of neutral lipids in adipose and non-adipose tissues. In addition, endothelium-dependent aortic relaxation is virtually abolished in ATGL-deficient aortas, indicating a severe endothelial dysfunction.In view of these findings, it was the aim of the present work to study the role of ATGL in NO/cGMP signal transduction. Human umbilical vein endothelial cells (HUVECs) and a microvascular endothelial cell line (HMEC-1) were treated with small interfering RNA (siRNA) targeted against ATGL. After incubation with siRNA for 24 h enzyme activity of endothelial NO synthase (eNOS) was measured by monitoring the conversion of [3H]L-arginine into [3H]L-citrulline. Additionally, eNOS phosphorylation at Serin-1177 and cGMP formation were determined by western blot analysis and radioimmunoassay, respectively.The results demonstrate that eNOS activity, cGMP formation and eNOS phosphorylation at Serin-1177 were not affected by ATGL knockdown. By contrast, preincubation with NM-221, an inhibitor of ATGL, led to a decrease in eNOS activity and cGMP formation, indicating a potential effect of ATGL on NO/cGMP signaling. However, the Zechner laboratory showed that NM-221 additionally affects lipolytic activity in an ATGL-independent way. Concerning these findings, the data obtained with NM-221 should be considered critically. Interestingly, prolonged incubation with siRNA (48 h and 72 h) resulted in decreased cGMP formation in endothelial cells treated with ATGL siRNA. These data suggest that longer periods of ATGL knockdown are required to determine possible effects of ATGL on NO/cGMP signaling. Further studies are necessary to confirm and clarify this issue.