c-Jun, Jun-B, and Jun-D proteins bind to the TPA response element (TRE) either as homodimers or as Jun-Fos heterodimers. We demonstrate that c-Jun and Jun-B nevertheless differ markedly in their ability to activate AP-1 responsive genes. c-Jun is an efficient activator of the c-jun and collagenase promoters, which contain a single TRE; Jun-B is not. Furthermore, Jun-B inhibits activation of these promoters by c-Jun. On the other hand, like c-Jun, Jun-B is an efficient activator of constructs containing multimeric TREs. Using chimeric proteins, we show that the distinct behavior of c-Jun and Jun-B is due to differences in their activation domains. Trans-activation by Jun-B depends on cooperative interactions between adjacently bound factors, while activation by c-Jun does not require such interactions. This differential behavior greatly expands the regulatory potential of the Jun family.