Rabbit liver DNA was cleaved with the restriction endonucleases Eco RI, Pst I or Kpn I. Resulting DNA fragments were denatured, separated by electrophoresis in agarose gels and subsequently transferred by blotting onto nitrocellulose filters. Filters were then hybridized with a plasmid containing a DNA copy of rabbit beta-globin messenger RNA (plasmid PbetaG1; Maniatis et al., 1976) which had been labeled by nick translation to a high specific activity with 32P. A very limited number of discrete rabbit DNA fragments was found which could form well matched hybrids with PbetaG1 DNA. Endonuclease Eco RI, which cleaves the rabbit beta-globin gene, produced two beta-globin DNA fragments, whereas endonucleases Pst I and Kpn I, which do not cut the gene, each generated only one fragment. Using double digests, these fragments could be ordered into a physical map of restriction endonuclease cleavage sites around the beta-globin gene on the rabbit genome. This map is consistent with the presence of a single copy of the beta-globin gene per haploid genome. The direction of transcription of the beta-globin gene within the physical map was determined.