Differential expression of the closely linked γ, β^A (or β^B), and β^C globin genes in sheep results in the production of fetal hemoglobin (Hb F, α₂γ₂) during gestation and the adult hemoglobins (Hb A, α₂β₂^A, and Hb B, α₂β₂^B) after birth. Erythropoietic stress in certain animals leads to production of Hb C (α₂β₂^C). The molecular mechanism of differential expression of these genes in nuclei of fetal and adult erythroid cells has been investigated by analysis of their susceptibility to digestion by DNase I (genes that are in the conformation associated with active transcription are sensitive to this nuclease). The concentration of globin gene sequences in DNA from control and DNase I-digested nuclei was determined by annealing to synthetic DNAs and analogous cDNA probes derived from recombinant plasmids containing one of the sheep globin genes. In nuclei from sheep fetal liver erythroid cells, the γ genes but not the β genes were digested by DNase I; the γ locus was open but the β^A or β^C loci was closed, consistent with synthesis of only Hb F by these cells. DNase I digestion of nuclei from bone marrow of anemic sheep making only Hb C or Hb B resulted in equivalent digestion of the β and γ gene sequences, although γ mRNA was not detected in these cells. Digestion by DNase I did not decrease the globin gene sequence concentration in residual DNA of spleen nuclei. As a further control, DNA from digested bone marrow and spleen nuclei were shown to anneal equally well to a cDNA prepared from liver polysomal mRNA. Differential expression of the γ and β globin genes in sheep fetal erythroid cell appears to be based on differences in chromatin structure. The γ globin gene remains in the active conformation in adult erythroid cells; failure of γ mRNA to accumulate in these cells probably reflects transcriptional or post-transcriptional regulation.