Activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) requires dimerization that is induced by phosphorylation of Tyr705, but its activity can be further modulated by phosphorylation at Ser727 in a manner that is dependent on cell context and the stimulus used. The role of STAT3 Ser727 phosphorylation in leptin signalling is currently not known. While cells transfected with the signalling-competent long form of the leptin receptor (ObRb) have been used to study leptin signalling, these are likely to be of limited use in studying STAT3 Ser727 phosphorylation due to the importance of cell background in determining the nature of the response. However, we have recently found that J774.2 macrophages endogenously express high levels of ObRb, and using these cells we find that leptin stimulates STAT3 phosphorylation on both Tyr705 and Ser727. The phosphorylation of Ser727 was not affected by rapamycin or the protein kinase C inhibitor H7 [1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride]. While the MEK-1 [mitogen-activated protein kinase (MAP kinase)/extracellular-signal-related kinase (ERK) kinase-1] inhibitor PD98059 [(2-amino-3'-methoxyphenyl)oxanaphthalen-4-one] had no effect on leptin-stimulated phosphorylation of STAT3 Tyr705, it greatly attenuated leptin's effects on STAT3 Ser727 phosphorylation. Further, Ob's effect on the DNA binding activity of STAT3 was also greatly reduced at all time points by PD98059. Leptin-induced ERK activation in J774.2 cells shows a biphasic pattern, with an initial reduction in ERK phosphorylation for up to 10 min following leptin stimulation, while at later time points phosphorylation of ERK was increased above basal levels. The increase in ERK activity corresponded with an increase in both phosphorylation of Ser727 and STAT3 DNA binding activity. These data provide the first evidence that ERK-mediated phosphorylation of Ser727 is required for full stimulation of STAT3 by leptin.