The p38 family of kinases is a subgroup of the mitogen-activated protein kinase family. It is composed of four isoforms and is involved in critical biological processes as well as in inflammatory diseases. The exact unique role of each p38 isoform in these processes is not understood well. To approach this question we have been developing intrinsically active variants of p38s. Recently we described a series of mutants of the human p38α, which were spontaneously active as recombinant proteins purified from Escherichia coli cells. We show here that some of these mutants are spontaneously active in several mammalian cells in culture. The spontaneous activity of some mutants is higher than the activity of the fully activated wild type counterpart. We further produced mutants of the other p38 isoforms and found that p38βD176A, p38γD179A, p38δ D176A, and p38δF324S are spontaneously active in vivo. The active mutants are also spontaneously phosphorylated. To test whether the mutants actually fulfill downstream duties of p38 proteins, we tested their effect on activating protein 1(AP-1)-mediated transcription. Active mutants of p38α induced AP-1-driven reporter genes, as well as the c-jun and c-fos promoters. An active variant of p38γ suppressed AP-1-mediated transcription. When active variants of p38α and p38γ were co-expressed, AP-1 activity was not induced, showing that p38γ is dominant over p38α with respect to AP-1 activation. Thus, intrinsically active variants that are spontaneously active in vivo have been obtained for all p38 isoforms. These variants have disclosed different effects of each isoform on AP-1 activity.