Natural deletions of the human gamma-globin gene cluster lead to specific syndromes characterized by increased production of fetal hemoglobin in adult life and provide a useful model to delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. A hypothesis accounting for these phenotypic features assumes that silencers located within the Agamma-to delta-gene region are deleted in hereditary persistence of fetal hemoglobin (HPFH) and deltabeta-thalassemias, leading to failure of switching. In the present study, we sought to clarify the in vivo role of two elements, termed Enh and F, located 3' to the Agamma-globin, in silencing the fetal genes. To this end, we generated three transgenic lines using cosmid constructs containing the full length of the globin locus control region (LCR) linked to the 3.3-kb Agamma-gene lacking both the Enh and F elements. The Enh/F deletion resulted in high levels of Agamma-globin gene expression in adult mice in all single copy lines, whereas, the LCR-Agamma single copy lines which retain the Enh and F elements exhibited complete normal switching of the fetal Agamma-gene. Our study documents directly for the first time the in vivo role of these two gene-proximal negative regulatory elements in silencing the fetal globin gene in the perinatal period, and thus these data may permit their eventual exploitation in therapeutic approaches for thalassemias.