Inhibition of lipolysis by palmitate, H2O2 and the anti-diabetic sulfonylurea drug, glimepiride, in isolated rat adipocytes has previously been shown to rely on the degradation of cyclic adenosine monophosphate by the phosphodiesterase, Gce1, and the 5'-nucleotidase, CD73. These glycosylphosphatidylinositol (GPI)-anchored proteins are translocated from plasma membrane lipid rafts to intracellular lipid droplets upon H2O2-induced activation of a GPI-specific phospholipase C (GPI-PLC) in response to palmitate and glimepiride in intact adipocytes and, as demonstrated here, in cell-free systems as well. The same agents are also known to stimulate the incorporation of fatty acids into triacylglycerol. Here the involvement of H2O2 production, GPI-PLC activation and translocation of Gce1 and CD73 in the agent-induced esterification and accompanying lipid droplet formation was tested in rat adipocytes using relevant inhibitors. The results demonstrate that upregulation of the esterification and accumulation of triacylglycerol by glimepiride depends on the sequential H2O2-induced GPI-PLC activation and GPI-protein translocation as does inhibition of lipolysis. In contrast, stimulation of the esterification and triacylglycerol accumulation by palmitate relies on insulin-independent tyrosine phosphorylation and thus differs from its anti-lipolytic mechanism. As expected, insulin regulates lipid metabolism via typical insulin signalling independent of H2O2 production, GPI-PLC activation and GPI-protein translocation, albeit these processes are moderately stimulated by insulin. In conclusion, triacylglycerol and lipid droplet formation in response to glimepiride and H2O2 may involve the hydrolysis of cyclic adenosine monophosphate by lipid droplet-associated Gce1 and CD73 which may regulate lipid droplet-associated triacylglycerol-synthesizing and hydrolyzing enzymes in coordinated and inverse fashion.