As mediators of cytokine and growth factor signaling, signal transducers and activators of transcription (STATs) transmit signals from the membrane and cytoplasm to the nucleus. While Y699 phosphorylation is required for STAT5b transcriptional activity, our previous studies show that mutation of two tyrosines in the transactivation domain of STAT5b (Y740/743F) increases Y699 phosphorylation leading to increased transcriptional activity and DNA synthesis in breast cancer cells [A.M. Weaver, C.M. Silva, Modulation of signal transducer and activator of transcription 5b activity in breast cancer cells by mutation of tyrosines within the transactivation domain, Molecular Endocrinology 20 (2006) 2392-2405]. In many instances, phosphorylation of serines in the transactivation domain also modulates STAT5b activity. Here, we demonstrate for the first time that EGF stimulation enhances S731 phosphorylation. Furthermore, we show that the increased activity of the Y740/743F STAT5b mutant requires S731. As STAT5b is implicated in several cancers, understanding how its activity is regulated through tyrosine and serine phosphorylation is vital for the development of potential novel cancer therapeutics. (C) 2007 Elsevier Inc. All rights reserved.