The actin regulatory protein cortactin is involved in multiple signaling pathways impinging on the cortical actin cytoskeleton. Cortactin is phosphorylated by ERK1/2 and Src family tyrosine kinases, resulting in neuronal Wiskott Aldrich Syndrome protein (N-WASp) activation and enhanced actin related protein (Arp)2/3-mediated actin nucleation. Cortactin migrates as an 80/85 kDa doublet when analyzed by SDS-PAGE. Phosphorylation by ERK1/2 is associated with conversion of the 80 kDa to the 85 kDa form, postulated to occur by inducing a conformational alteration that releases the carboxyl-terminal SH3 domain from autoinhibition. Our recent analysis of the 80-85 kDa cortactin "shift" in tumor cells indicates that while ERK1/2 phosphorylation is associated with the 85 kDa shift, this phosphorylation event is not required for the shift to occur, nor does ERK1/2 phosphorylation appreciably alter global cortactin confirmation. These data indicate that additional factors besides ERK1/2 phosphorylation contribute to generating and/or maintaining the activated 85 kDa cortactin form in stimulated cells.