Recent studies indicate that genes from only one nucleolus organizer undergo replication during polytenization of salivary gland cells of X/Y flies. This report presents evidence that this is also true X/X polytene cells. Interstrain hybrids were constructed which contain X chromosomes with different rDNA hybridization patterns following digestion with Eco RI, and DNA from diploid or polytene tissue was analyzed using Southern transfers. DNA from diploid tissue of X/X hybrids shows rDNA patterns from both X chromosomes, while DNA from polytene tissue of hybrids shows a pattern characteristic of only one of the X chromosomes. The polytene pattern of the hybrid is independent of whether the dominant X chromosome was derived from the female or male parent. These results suggest that genes from only one nucleolus organizer undergo multiple rounds of replication in X/X polytene cells, and account for the finding that X/O and X/X polytene cells contain similar levels of rDNA without postulating compensation as a ribosomal gene rectification mechanism.