Expression of immediate-early genes involving the 12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive element (TRE) is modulated by post-translational modification of pre-existing activator protein 1 (AP-1) constituents. One of the components of AP-1, c-Jun, has been shown to be phosphorylated by glycogen synthase kinase 3 (GSK-3) in vitro in a region proximal to the DNA-binding domain, resulting in decreased DNA binding. Here, we have used transient transfection to show that AP-1 activity is inhibitable by coexpression of GSK-3 in intact cells. Furthermore, we show that the c-Jun-related proteins JunD and JunB are subject to similar regulation by GSK-3 in intact cells. Comparison of tryptic phosphopeptide maps of the three Jun proteins incubated with GSK-3 in vitro with maps of the same proteins immunoprecipitated from 32P-labelled cells indicates similar sites of phosphorylation. Together, these data support the hypothesis that GSK-3 is an important regulator of AP-1 activity in vivo.