The TOJ3 gene was originally identified on the basis of its specific activation in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17). Overexpression of TOJ3 induces cellular transformation of embryonic avian fibroblasts, revealing an intrinsic oncogenic potential. Transforming activity has also been demonstrated for MSP58, the human homolog of TOJ3, and oncogenic cell transformation by MSP58 is specifically inhibited by the tumor suppressor PTEN. To investigate the mechanism of aberrant TOJ3 gene activation in jun-transformed fibroblasts, the entire quail TOJ3 gene including 13 exons and the 5' regulatory region was isolated. Functional analyses of the promoter by transcriptional transactivation assays revealed that the specific induction of TOJ3 is mediated by a cluster of three noncanonical AP-1 binding motifs (5'-CAGCTCA-3' or 5'-CACCTCA-3') which share the 3' half-site with the consensus motif (5'-TGA(C)/(G)TCA-3'). Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses showed that Jun binds to these motifs with an affinity similar to that observed for binding to an AP-1 consensus site. Noncanonical binding sites are also present in the chicken and human TOJ3/MSP58 promoter regions. These results confirm and extend the previous observation that TOJ3 represents an immediate effector gene of Jun and may point to an essential role of TOJ3/MSP58 in carcinogenesis involving aberrant AP-1 expression.