A detailed restriction map was determined for a 10.9 KB region that contains the initiation site for 45S pre-rRNA and the first 1.7 KB of the 18S rRNA coding region. When the restriction pattern of the cloned rDNA was compared with that of total rat DNA, the rDNA regions of both Sprague-Dawley and BD-9 rats were identical to each other and to that of the cloned rDNA. However, both strains exhibit a major polymorphism consisting of an insertion of 0.9 KB of DNA in the nontranscribed spacer between 0.29 KB and 1.8 KB upstream from the 45S RNA initiation site. This region consists of tandem repeats approximately 130 base pairs in length. These repeats contain large poly T tracts and are similar in sequence to analogous elements 5' to the origin of mouse rRNA transcription. Regions containing highly repetitious DNA sequences were located at sites 2.8 KB and 4.3 KB upstream from the initiation site. The repetitive sequence at 2.8 KB from the initiation site anneal to a known Alu-equivalent type 2 sequence derived from the second intron of the rat growth hormone gene.