Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21WAF1 is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21WAF1 mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21WAF13′-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21WAF1 3′-UTR constructs identified multiplecis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5′ region of the p21WAF1 3′-UTR, whereas EGF induced reporter activity in constructs containing sequences 3′ of the UVC-responsive region. Thesecis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21WAF1mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21WAF1 protein expression or growth inhibition. However, binding of HuR to its target 3′-UTRcis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21WAF1mRNA via different mechanisms. We conclude that EGF-induced p21WAF1 protein expression is mediated largely by stabilization of p21WAF1 mRNA elicited via multiple 3′-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21WAF1 expression or growth inhibition in this system. CP1 is a novel p21WAF1 mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21WAF1 mRNA stability.