Drosophila circadian clock gene timeless (tim) displays circadian oscillation in its mRNA level, and such oscillation is transcriptionally regulated. The promoter region up to -756 of tim is suggested to promote the circadian mRNA expression, however, the role of the sequence upstream of tim promoter region in the transcriptional regulation is still unrevealed, We novelly isolated and determined tim 5'-flanking sequence -2764 to -757, and found a putative cAMP-response element, six regions of the half site for PAR-basic leucine zipper transcription factors and six nonpalindromic E-boxes. Our in vivo reporter assay showed that 966 bp of tim promoter region, including a palindromic CACGTG E-box and a half site of PAR-basic leucine zipper transcription factors, was minimally required for its daily mRNA expression. While, the deletion of the 5'-flanking region -1970 to -967 caused a slight decrease in the reporter mRNA levels. These results indicate that the 5'-flanking sequence upstream of the promoter region have a role in the daily regulation of tim mRNA expression in Drosophila.