The c-myc oncogene has been implicated in the genesis of tumors as it has been found to undergo rearrangements and structural alterations in several types of neoplasms. However, the molecular mechanism of its involvement in transformation is still unclear as is the function of its protein product. We have studied the biological activity of the normal human c-myc oncogene in a well-characterized rat fibroblast line, F2408. The human gene, deleted of its 5' non-coding exon, was placed under the control of the Polyoma virus DNA regulatory region, linked to a dominant selectable marker (neomycin-resistance gene) and transfected into F2408 cells. Several neoR colonies were assayed for ability to grow in agar suspension, and the steady-state levels of mRNA transcripts of the exogenous c-myc gene were analyzed by Northern blot. While the greatest majority of these cell populations are phenotypically normal and express low levels of human myc transcripts, the rare neoR clones which display anchorage-independence express the human gene at high levels. This correlation was confirmed by the analysis of two neoR lines which contain low levels of c-myc RNA but gave rise to agar colonies at a frequency of about 10(-5). These agar derivatives were found to have greatly increased levels of c-myc mRNA when compared to the respective parental lines. The clones expressing high levels of myc RNA were found to be tumorigenic upon inoculation into young syngeneic rats. These results suggest that constitutive high expression of the normal c-myc gene is sufficient to confer anchorage-independence and tumorigenic potential to established rat fibroblasts.