A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [125I]18S or [125I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5 x 10(6) daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.