The constitutive activation of nuclear factor kappaB (NFkappaB) helps a variety of tumors to resist apoptosis and desensitizes them to chemotherapy, but the causes are still largely unknown. We have analysed this phenomenon in eight mutant cell lines derived from human 293 cells, selected for NFkappaB-dependent expression of a marker gene, and also in seven tumor-derived cell lines. Conditioned media from all of these cells stimulated the activation of NFkappaB (up to 30-fold) in indicator cells carrying an NFkappaB-responsive reporter. Therefore, secretion of extracellular factors as the cause of constitutive activation seems to be general. The mRNAs encoding several different cytokines and growth factors were greatly overexpressed in the tumor and mutant cells. The pattern of overexpression was distinct in each cell line, indicating that the phenomenon is complex. Two secreted factors whose roles in the constitutive activation of NFkappaB are not well defined were investigated further as pure proteins: transforming growth factor beta2 (TGFbeta2) and fibroblast growth factor 5 (FGF5) were both highly expressed in some mutant clones and tumor cell lines, each activated NFkappaB alone, and the combination was synergistic. Our data indicate that a group of different factors, expressed at abnormally high levels, can contribute singly and synergistically to the constitutive activation of NFkappaB in all of the mutant and tumor cell lines we studied. Since several NFkappaB target genes encode secreted proteins that induce NFkappaB, autocrine loops are likely to be ubiquitously important in the constitutive activation of NFkappaB in cancer. We provide the first evidence of the general, complex, and synergistic activation of NFkappaB in tumor and mutant cell lines through the action of secreted factors and suggest that the same explanation is likely for the constitutive activation of NFkappaB in cancers.