Human ribosomal DNA has been inferred to be organized in tandem repeat units of 44 kb, of which only 13 kb is transcribed into preribosomal RNA. Unfortunately, it has remained difficult to examine the intact repeat structure directly, because even a single repeat unit is too large to be accommodated in conventional cloning systems. Here we report the isolation of intact repeat units using yeast artificial chromosomes as a cloning tool. With a spacer sequence specific to human ribosomal DNA used as a probe, 27 clones were identified among 17,000 YACs (about 0.7 genomic equivalent of total human DNA). Fourteen clones contained only a small portion of rDNA; the other 13 contained most or all of the rDNA repeat unit, and 8 of those were studied in further detail. They contained 1 to 1.5 repeat units of rDNA with all of the expected EcoRI and HindIII fragments. These clones provide possible starting material for the analysis of expression of a single unit of rDNA. Unexpectedly, however, only the four smaller clones (70 to 90 kb) were completely composed of standard rDNA sequences; four larger clones (up to 950 kb in length) contained additional "non-rDNA" sequences, at either one or both ends of the repeat unit. Analysis of these atypical rDNA clones suggests that their inserts either are scattered in the genome or are localized in a nucleolar organizer region that is more complex than previously recognized.