The chicken karyotype consists of 39 chromosomes of which 33 are classed as microchromosomes (MICs). MICs contain about one third of genomic DNA. The majority of mapped chicken genes are assigned to macrochromosomes (MACs), but a recent study indicated that CpG islands (CGIs), which are associated with most vertebrate genes, map predominantly to MICs. The present work establishes that chicken genes are concentrated on MICs by several criteria. Acetylated (lysine 5) histone H4, which is strongly correlated with the presence of genes, is highly enriched on MICs by immunocytochemistry. In addition, detailed analysis of chicken cosmids shows that CGI-like fragments are approximately six times denser on MICs than on MACs. Published mapping of randomly chosen genes by fluorescent in situ hybridization (FISH) also shows a significant excess of microchromosomal assignments. Finally, the finding that MICs replicate during the first half of S phase is also compatible with the suggestion that MICs represent gene-rich DNA. We use the cosmid data to predict that approximately 75% of chicken genes are located on microchromosomes. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AJ001643 and AJ001644.]