The human beta-globin locus control region (LCR) is composed of four erythroid-specific, DNase I hypersensitive (HS) sites that are located 6 to 18 kb upstream of the epsilon-globin gene. The beta-globin LCR appears to have two major functions. First, the sequences "open" a chromosomal domain that includes the epsilon-, gamma-, and beta-globin genes and, second, the LCR directs high-level, erythroid-specific expression of each globin gene family member. An LCR subfragment containing only 5' HS 2 can confer these properties on a linked beta-globin gene. To determine whether 5' HS 2 can form an erythroid-specific, DNase I hypersensitive site in the absence of a linked globin gene, a 1.9-kb DNA fragment containing this site was injected into fertilized mouse eggs and DNase I hypersensitivity was analyzed in the animals that developed. In 9 of 10 transgenic mouse lines, the human 5' HS 2 fragment formed a DNase I hypersensitive site in fetal liver but not in fetal brain. These results suggest that human 5' HS 2 can function autonomously to organize an open chromatin domain specifically in erythroid cells.