To understand the mechanism by which the Myc protein contributes to cell growth and development, our laboratory is studying functions of the avian myelocytomatosis virus 29 (MC29) Gag-Myc protein (v-Myc) in the mouse fibroblast cell line C3H10T1/2. Previously, we identified two specific regions in v-Myc which are required for co-transformation with activated H-ras. One maps to the amino-terminal portion of v-Myc (amino acids 1-137) and has the potential to activate transcription of a basal promoter. The second region spans the carboxy-terminal region of v-Myc (amino acids 244-410), contains a basic/helix-loop-helix/leucine zipper motif and specifies the nuclear location of the protein. In this study, we have generated a series of deletion mutations within the MC29 gag-myc gene to define precisely the carboxy-terminal transforming region using the co-transformation of C3H10T1/2 cells as an assay. v-Myc proteins encoded by selected deletion mutations were also examined for their intracellular location, the ability to interact with the Max protein and the potential to bind specifically to DNA. Our results demonstrate that integrity of both the basic/helix-loop-helix and the leucine zipper motifs of v-Myc is required for co-transforming activity, but that the major nuclear localization signal sequence of v-Myc can be deleted without compromising the ability of v-Myc to cooperate with activated H-Ras p21 to transform C3H10T1/2 cells. In addition, while the binding of v-Myc to Max is necessary for ras/myc co-transformation, it is not sufficient, and also requires the integrity of Myc sequences specifying site-specific DNA binding.