We have identified a novel c-Myc responsive gene, named JPO1, by representational difference analysis. JPO1 responds to two inducible c-Myc systems and behaves as a direct c-Myc target gene. JPO1 mRNA expression is readily detectable in the thymus, small intestine and colon, whereas expression is relatively low in spleen, bone marrow, and peripheral leukocytes. We cloned a full length JPO1 cDNA that encodes a 47 kDa nuclear protein. To determine the role of JPO1 in Myc-mediated cellular phenotypes, stable Rat1a fibroblasts over-expressing JPO1 were tested and compared to transformed Rat1a-Myc cells. Although JPO1 has a diminished transforming activity as compared with c-Myc, JPO1 complements a transformation-defective Myc Box II mutant in the Rat1a transformation assay. This complementation provides evidence for a genetic link between c-Myc and JPO1. Similar to c-Myc, JPO1 over-expression enhances the clonogenicity of CB33 human lymphoblastoid cells in methylcellulose assays. These observations suggest that JPO1 participates in c-Myc mediated transformation, supporting an emerging concept that c-Myc target genes constitute nodal points in a network of pathways that lead from c-Myc to various Myc-related phenotypes and ultimately to tumorigenesis.