The genes coding for the 18S and 28S rRNAs in D. melanogaster were examined using Southern transfers of DNA from diploid or polytene tissue. A ribosomal gene repeat 12 kb in length is present in DNA from diploid tissue of males and is the major repeat on the Y chromosome. This repeat is present in low amounts on the X chromosome, which contains major repeats of 17 and 11.5 kb. In polytene nuclei of males, the 12 kb band is disproportionately replicated, and only a very low amount of the 11.5 kb repeat and no 17 kb repeat are detected. Polytene nuclei of females contain reduced amounts of the 17 kb repeat relative to the 11.5 kb repeat. This disproportionate replication of specific ribosomal gene repeats suggests that polytenization of the rDNA may involve an extrachromosomal mechanism. Evidence that genes from only one nucleolus organizer are replicated during polytenization in X/Y and X/X flies is discussed. A method for analyzing DNA from tissue of individual larvae was developed to test for population heterogeneity in ribosomal gene structure. Heterogeneity was observed in the ribosomal genes of three Ore R lines, four other D. melanogaster strains and between males and females of the same strain.