Ribosomal DNA (rDNA) from calf was isolated by three density gradient centrifugations. The first centrifugation in Cs2S04/BAMD was used to obtain partially resolved dG+dC-rich fractions from total DNA. The second and third centrifugations, in Cs2S04/Ag+, led to the isolation of an rDNA fraction characterized by a symmetrical band in CsCl, p = 1.724 g/cm3. This new procedure appears to be generally suitable for the isolation of rDNA and other dG+dC-rich repeated genes. The organization of isolated calf rDNA has been studied by restriction enzyme digestion and by hybridization with cloned rDNA from Xenopus laevis. The repeat unit of calf rDNA has a molecular weight of 21x10(6) and is split by EcoR1 into two fragments, 16x10(6) and 5.0x10(6), and by BamHI into seven fragments. EcoRI and BamHI sites have been mapped. Most of the 18S and 28S RNA genes and the transcribed spacer are contained in the small EcoRI fragment, while the non-transcribed spacer is localized in the large EcoRI fragment. This spacer showed length heterogeneity within a single individual; such heterogeneity is limited to two regions of the spacer.