Many growth factors, including epidermal growth factor (EGF), can activate the signal transducer and activator of transcription (STAT) signaling pathway. Here, we demonstrate that STAT activation by EGF treatment is conditional. EGF activates STAT1 and STAT3 in A431 but not in HeLa and PC12 cells. Using a reconstituted in vitro STAT activation system, we have identified and partially purified a potential inhibitor (s) that is membrane- associated and can block STAT activation induced by EGF in vitro. However, this inhibitor has no effect on STAT complexes after they are formed. We have further shown that this inhibitor(s) also exists in many other cancer cell lines, suggesting that blocking the STAT activation during growth factor signal transduction may play a significant role in the development of many kinds of cancers.