Dimerization plays a pivotal role in modulating the activity of the c-Jun proto-oncogene product. Heterodimerization with activating transcription factor-2 (ATF-2) alters the DNA-binding specificity of c-Jun, allowing its targeting to several cAMP responsive element (CRE)-related sequences, which control a subset of AP-1-responsive genes. Here we show that a c-Jun/ATF-2 heterodimer binds to the AP-1 site (uPA 5'-TRE) essential for the activity of the human urokinase enhancer, conferring on this element several distinctive regulatory properties. The c-Jun/ATF-2 heterodimer was identified by binding competition assays, u.v. cross linking, and monospecific antibodies. In vitro binding studies revealed that the uPA 5'-TRE sequence is recognized by the cyclic AMP-unresponsive ATF-2 factor, but not by the cyclic AMP-inducible CREB. In addition, in vivo studies suggest that ATF-2 can mediate, at the same time, the activation of the c-Jun/ATF-2 site and the repression of the canonical collagenase AP-1 site. We report that heterodimerization with c-Fos does not increase the binding of c-Jun to the uPA 5'-TRE, in contrast to the increased binding at a consensus AP-1 site. Our data further suggest that c-Fos can act as a repressor of the c-Jun/ATF-2 binding site, revealing an important functional difference, with respect to canonical AP-1 elements.