The Drosophila melanogaster ribosomal DNA (rDNA) functions as an X-Y meiotic pairing site. Deletions encompassing the X chromosomal rDNA block (located in the heterochromatin) disrupt X-Y pairing and disjunction. Insertions of single, complete rRNA genes at ectopic locations on the heterochromatically deficient X partially restore X-Y pairing capacity. This study was undertaken to test fragments of an rDNA repeat for the ability to stimulate X-Y pairing and disjunction and to test for relationships between pairing capacity and two other phenotypes associated with rDNA insertions: transcription and the ability to organize a nucleolus. Insertions of three different fragments, all of which retained the rDNA promoter and upstream spacer sequences and which differed among each other in the length of downstream sequences, were obtained by P-element mediated transformation. One of the fragments is truncated only 140bp downstream from the promoter. Insertions of all three fragments proved capable of stimulating X-Y disjunction. Double insertions were substantially more effective than single insertions. RNA/PCR analysis was used to show that transcripts initiated at the inserted rDNA promoters are present in testis RNA from all insertions. Treatment with an antinucleolar antibody revealed that none of the insertions was associated with a mininucleolus. Thus promoter-containing rDNA fragments are autonomously capable of being transcribed and of functioning as X-Y pairing sites, but not of forming a mini-nucleolus.