Tumors induced in vivo by v-myc recombinant retroviruses and mouse cell lines from a variety of lineages infected with these viruses, which express high levels of avian v-myc, were found to be invariably associated with a lack of c-myc expression. In order to distinguish between v-myc-induced shutdown versus a differentiation-associated down regulation of c-myc expression, we have determined steady state levels of c-myc mRNA in three different cell lines in culture which express various levels of c-myc prior to infection. Extreme levels of v-myc expression (10- to 100-fold excess over c-myc) was achieved in a myeloid (FDC-P1) and a T lymphoid (CTB-6) cell line. In both lines, c-myc expression was absent in the infected cells as assessed by RNA and nuclear run-on analyses and, in the case of FDC-P1, could not be induced by growth factor (IL-3) or inhibitors of protein systhesis (to remove a labile repressor). Moreover, DNase I hypersensitive sites typical for active c-myc alleles were absent in FDC-P1 cells infected with v-myc retroviruses. In NIH 3T3 fibroblast cells, v-myc was expressed at least five times the levels of c-myc present in uninfected cells. Again, v-myc expression was associated with down-regulation of c-myc. However, in contrast to infected FDC-P1 cells, down-regulated NIH 3T3 cells could be induced for c-myc expression by treatment with anisomycin for periods corresponding to greater than three times the half-life of v-myc protein in these cells. A deletion analysis of v-myc revealed that removal of 109 amino acids from the beginning of the sequence derived from the second c-myc coding exon was sufficient to abolish its ability to down-regulate c-myc expression.