Previous experiments have demonstrated that the human β-globin gene is correctly regulated in transgenic mice. The β-globin gene is not expressed in yolk sac-derived erythroid cells in early embryonic development but is expressed concomitantly with the adult mouse β-globin genes in 14- to 16-day fetal liver and adult reticulocytes. In an attempt to localize sequences that direct erythroid-specific expression, fragments of the human β-globin gene were inserted in the opposite orientation 200 base pairs (bp) upstream of an intact human (A)γ marker gene, which is not expressed on its own in mouse fetal liver. In the experiments reported here, two β-globin 3' sequences activated the marker gene specifically in fetal liver. One sequence is located in a 250-bp Pst I fragment 550-800 bp downstream from the poly(A) site; the other is located near an EcoRI site in the third exon. These two sequences are active individually, and their combined effect is greater than their effects alone. β-Globin 5' sequences from -815 to -50 were also analyzed for activity in this assay. The 5' sequences did not activate the marker gene when tested alone but did stimulate expression that was already directed to adult erythroid tissue by the two 3' sequences. These results suggest that three separate sequences are involved in human β-globin gene regulation. The two 3' sequences act as adult erythroid enhances and the 5' sequence stimulates expression that is already determined to be erythroid specific.