Increasing evidence supports an important biological role for Myc in the downregulation of specific gene transcription. Recent studies suggest that c-Myc may suppress promoter activity through proteins of the basal transcription machinery. We have previously reported that Myc protein, in combination with additional cellular factors, suppresses transcription initiation from the c-myc promoter. To characterize the cis components of this Myc negative autoregulation pathway, fragments of the human c-myc promoter were inserted upstream of luciferase reporter genes and assayed for responsiveness to inducible MycER activation in Rat-1 fibroblasts. We found four- to fivefold suppression of a c-myc P2 minimal promoter fragment upon induction of wild-type MycER protein activity, while induction of a mutant MycER protein lacking amino acids 106 to 143 required for Myc autosuppression failed to elicit this response. This assay is physiologically significant, as it reflects Myc autosuppression of the endogenous c-myc gene with regard to kinetics, dose dependency, cell type specificity, and c-Myc functional domains. Analysis of mutations within the P2 minimal promoter indicated that the cis components of Myc autosuppression could not be ascribed to any known protein-binding motifs. In addition, to address the trans factors required for Myc negative autoregulation, we expressed MycEG and MaxEG leucine zipper dimerization mutants in Rat-1 cells and found that Myc-Max heterodimerization is obligatory for Myc autosuppression. Two models for the Myc autosuppression mechanism are discussed.