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Tandem repeats of ribosomal RNA transcription units in Drosophila melanogaster are separated by a nontranscribed spacer that is comprised in part of serial repeats of a 0.24 kb sequence. DNA sequence analysis shows that such repeats are imperfect copies of a region that includes the site of in vivo rRNA transcription initiation (ca. -240 to +30). Subclones of the rDNA spacer that are copies of the sequence extending from -34 through the initiation site support detectable in vitro transcription in a mixture involving a Drosophila cell-free extract, but accurate in vitro transcription is considerably enhanced when a nontranscribed spacer template includes a copy of the sequence extending upstream of -34. From a comparison of the sequence and transcription template-effectiveness of various rDNA subclones, we infer that a major promoter of RNA polymerase I activity lies between -150 and -30 in the rDNA nontranscribed spacer. The nontranscribed spacer copies of the initiation region are less effective templates for transcription than is the region of in vivo initiation and there are differences between spacer repeates and the authentic sequence downstream of -240 that may account for this. © 1982 IRL Press Limited.