We have cloned a human genomic DNA fragment that contains a complex bent DNA structure and a LINE-1 (L1) sequence. The bent DNA structure spans from 70 to -440 relative to the transcription start site of L1, and contains a left-handed sup erhelical structure from about 70 to -200. When we changed the rotational orientation of the bent DNA relative to the L1 promoter by inserting double-stranded oligonucleotides between positions -3 and -4, L1 promoter activity was profoundly affected Inserts of 5 and 16 by altered the rotational position by about 180°, and stimulated transcription 2- to 3-fold Insertions of 11 and 21 base pairs, which altered the rotational orientati on minimally, had much less effect. We present here the first experimental evidence that upstream DNA architecture influences the machinery employed in L1 transcription.