Myc proteins function in heterodimeric complexes with Max proteins as transcriptional regulators at least in part by binding to E box sequences with a 5'-CACGTG core. Since such E boxes are found frequently in the human genome and since other proteins besides Myc/Max can bind to similar or identical sequences it is unclear how the specificity of E box-mediated gene transcription is determined. Recent findings were interpreted to suggest that Myc/Max, but not Max/Max or USF complexes, bind cooperatively to DNA sequences that contain two E box elements. This provides a potential mechanism for selective E box-mediated gene transcription. To extend this finding we analyzed DNA binding of c-Myc/Max complexes using a transient COS-7 expression system. In both competition and titration experiments no cooperative binding of c-Myc/Max heterodimers to probes with two E boxes was observed. Furthermore, c-Myc-specific transcription of reporter gene constructs did not reveal cooperativity. Thus ourfindings argue against cooperative DNA binding of Myc proteins as a selection mechanism for E box-dependent transcription.