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The chicken adult beta-globin gene is one of the more intensively investigated developmentally regulated loci in higher eukaryotes. Detailed molecular analysis of the locus allows precise examination of the chromosomal changes that occur on activation of the gene during erythroid maturation. The best studied of these changes are the acquisition of DNase I hypersensitivity, developmentally correlated alteration of CpG-specific cytosine methylation patterns and in vitro assembly of erythroid-specific protein complexes 5' to the gene that mimics in vivo creation of the 5' DNase I hypersensitive 'region' lying 60 to 260 nucleotides 5' to the beta-globin cap site in red blood cell chromatin. Here we demonstrate that proximal beta-globin DNA sequences lying greater than 112 base pairs (bp) 5' to the cap site are not involved in determining the erythroid-specific induction characteristics of this gene in transient expression assays, whereas an enhancer sequence within a 300-bp PvuII fragment lying approximately 400 nucleotides 3' to the polyadenylation signal is intimately involved in determining the erythroid cell specificity and correct time of induction of beta-globin transcription during red cell maturation.