We have analyzed the chromosome structure and expression of the alpha-gene cluster in developing chicken embryos. Using recombinant lambda clones (Dodgson and Engel, 1980), we show a striking relationship between chromosomal regions that are unmethylated, regions that are DNAase I-sensitive and regions that are transcribed. Adjacent regions at both the 5' and 3' sides of the active alpha genes are methylated and relatively insensitive to DNAase I. The active alpha subdomain defined by these assays begins right at the 5' side of the first alpha gene (alpha D) in the active cluster in definitive red cells and extends through a 1.5 kb spacer, into the second alpha gene (alpha A), and 1.5 kb beyond the 3' side of that gene. The sharp boundaries of this subdomain suggest that specific DNA sequences may establish its borders. The extension of the active chromosomal domain beyond the most stable nuclear transcript suggests that transcription may proceed beyond the 3' ends of both alpha A and alpha D. This has been verified by in vitro runoff nuclear transcription. Presumably, poly(A) addition occurs before transcription is terminated. During the switch from the primitive to definitive lineage of erythroblasts, the so-called U gene becomes inactive. This inactivity is reflected in its assembly into a more DNAase-resistant structure. The associated DNA also becomes methylated, and no transcription is detectable by endogenous RNA polymerases. A DNAase I-hypersensitive region at the gene becomes inactivated after the switch to the definitive lineage.