In situ hybridization of sea urchin (Psammechinus miliaris, Lytechinus pictus and Strongylocentrotus purpuratus) histone messenger RNA has been used to map complementary sequences on polytene chromosomes from Drosophila melanogaster. The sea urchin RNA hybridizes to the polytene regions from 39D3 through 39E1-2, including both of these bands (39D2 may also be included). This region is identical to the one which hybridizes most heavily with non-polyadenylated cytoplasmic RNA from D. melanogaster tissues. Sea urchin mRNAs coding for several individual histones each hybridize across the entire region from 39D3 (or D2) through 39E1-2, as would be expected if the individual mRNA sequences are interspersed. In view of the apparently even distribution of sequences complementary to histone mRNA within the 39D3-39E1-2 region, the significance of the several polytene bands in this region remains an open question. Biochemical characterization of the hybrids between sea urchin histone mRNA and D. melanogaster DNA suggest that sea urchin mRNAs for several of the histone classes have some portions which retain enough sequence homology with the D. melanogaster sequences to form hybrids, although the hybrids have base pair mismatches. In situ hybridization of chromosomes in which region 39-E is ectopically paired show no evidence of seqence homology in the chromosome region with which 39D-E is associated.