Abstract Previous study has demonstrated that a far upstream 174-bp spacer sequence of the rat rRNA-encoding (rDNA) gene can function as an enhancer in vitro in an orientation- and distance-independent manner [Dixit et al. J. Biol. Chem. 262 (1987) 11616–11622]. To demonstrate that this element can also function in vivo, two rat rDNA- cat plasmids, one with the 174-bp element and the other without this sequence, were constructed and transfected into CHO cells. Primer extension analysis of the transcripts produced after transfection showed that transcription initiation occurred at the +1 site of the rDNA. The 174-bp sequence stimulated the rat poll promoter activity in cis 4–5-fold over the control (with the promoter alone). This RNA polymerase (poll) enhancer also stimulated the mouse metallothionein-I ( MT-I) and SV40 promoter activities in vivo, irrespective of its distance and orientation. Further dissection of the 174-bp element revealed that the stimulatory activity on the RNA polymerase II (polII) promoter resides within the 37-bp and 43-bp domains at the 3' end of the 174-bp element. Unlike this spacer enhancer, the 130-bp repeat element ( RE) proximal to the rat promoter [Ghosh et al., Gene 125 (1993) 217–222] was unable to modulate the polII promoter activity in vivo. These data show that while the non-repetitive enhancer sequence of rat rDNA is interchangeable for the poll and polII promoters, the RE is poll-specific.