Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on the GCR1 product, a transcription factor which also regulates the genes encoding yeast glycolytic enzymes. Eliminating GCR1 causes a 100-fold decrease in transcription of Ty2-917. Activation by the isolated Ty2-917 UAS also strongly depends on GCR1. Unexpectedly, GCR1-dependent activation by the Ty2-917 enhancer is strongly position-dependent. Activation by the enhancer in its normal position within the transcription unit depended strongly on GCR1, but eliminating GCR1 reduced activation only three-fold when the enhancer was moved upstream of the transcribed region. Gel mobility shift and DNaseI protection assays indicated that GCR1 binds specifically to multiple sites within the Ty2-917 UAS and enhancer regions. (C) 1997 by John Wiley & Sons, Ltd.