The 5'-upstream region of the small nuclear ribonucleoprotein E protein (snRNP E) gene has multiple sequence similarities to elements found to be integral in the transcriptional regulation of some mouse ribosomal protein genes (G + C block, CTTCCG motifs) as well as U1 snRNA genes (U1 "B," U1 "D," and SPH enhancers). Furthermore, the immediate upstream region of the snRNP E protein gene lacks typical TATA and CAAT sequence motifs but contains one copy of a GC box characteristic of a housekeeping promoter. By transfection of constructs containing various amounts of the 5'-upstream region of the snRNP E protein gene linked to the bacterial gene for chloramphenicol acetyl transferase (CAT), we determined that the 5' boundary of sequence needed for transcription was within the first 153 nucleotides upstream of the transcription start site. Further deletions to within 51 base pairs reduced CAT expression by 2.5-fold. Mutational analysis within this 51-base pair region revealed that direct repeats of the hexamer CTTCCG were essential for CAT expression. Gel shift assays and DNase I footprinting experiments confirmed this conclusion by demonstrating specific binding of proteins to the CTTCCG motifs. This suggests that the direct repeats of the CTTCCG hexamer sequence play a key role in transcriptional regulation of the snRNP E protein gene.