Basic-Helix-Loop-Helix-Per-Arnt-Sim (bHLH-PAS) transcription factor, Brain-Muscle-Arnt-Like-protein 1 (BMAL1), forms a heterodimer with the CLOCK protein. The BMAL1/CLOCK complex binds to a specific DNA sequence and plays an essential role in the generation of the circadian rhythm. The basic region of BMAL1 contains an E-R-X-R motif that is highly conserved among basic-helix-loop-helix (bHLH) transcription factors that bind to the E-box transcription element, and is thus thought to constitute a structure required for recognition of this DNA sequence. To understand the role of arginine 91 (E-K-R-R) within the basic region of BMAL1, we examined the effect of mutation of this residue on BMAL1 function. Co-immunoprecipitation and electrophoretic mobility shift assays (EMSA) showed that a BMAL1 R91A mutant forms a heterodimer with CLOCK, but is unable to support DNA binding in vitro. Consistent with these observations, plasmids encoding the R91A and R91H mutants of BMAL1 were unable to stimulate transcription from an E-box containing reporter construct in transient co-transfection analyses in NIH 3T3 cells. More importantly, these mutants suppressed the activation of transcription from the reporter construct mediated by wild-type BMAL1, indicating that they possess dominant negative activity in this assay. These results document further the importance of the basic region of BMAL1 in binding to DNA and suggest that this new mutant might be a useful tool for further analysis of BMAL1 function.