The GATA-1 gene encodes a transcription factor expressed in early multipotent haemopoietic progenitors, in more mature cells of the erythroid, megakaryocytic and other lineages, but not in late myeloid precursors; its function is essential for the normal development of the erythroid and megakaryocytic system. To define regulatory elements of the mouse GATA-1 gene, we mapped DNaseI-hypersensitive sites in nuclei of erythroid and haemopoietic progenitor cells. Five sites were detected. The two upstream sites, site 1 and site 2, represent a new and a previously defined erythroid enhancer respectively. The site 1 enhancer activity depends both on a GATA-binding site (also footprinted in vivo) and on several sites capable of binding relatively ubiquitous factors. A DNA fragment encompassing site 1, placed upstream of a GATA-1 minimal promoter, is able to drive expression of a simian virus 40 (SV40) T-antigen in the yolk sac, but not bone-marrow cells, obtained from mice transgenic for this construct, allowing in vitro establishment of immortalized yolk-sac cells. A similar construct including site 2, instead of site 1, and previously shown to be able to immortalize adult marrow cells is not significantly active in yolk-sac cells. Sites 4 and 5, located in the first large intron, have no enhancer activity; they include a long array of potential Ets-binding sites. MnlI restriction sites, overlapping some of the Ets sites, are highly accessible, in intact nuclei, to MnlI. Although these sites are present in all GATA-1-expressing cells studied, they are the only strong sites detectable in FDCP-mix multipotent progenitor cells, most of which do not yet express GATA-1. The data indicate that appropriate GATA-1 regulation may require the co-operation of different regulatory elements acting at different stages of development and cell differentiation.