Previous studies have shown that a ‐ 112 to + 78 DNA fragment from the erythroid promoter of the human porphobilinogen deaminase (PBGD) gene has erythroid‐specific activity. This PBGD‐(‐112 to + 78) promoter contains a CCACC binding site (position ‐ 100), a GATA binding site (position ‐ 70) and an initiator element around the cap site. Using a cotransfection assay, we find that the human factor GATA‐1 trans ‐activates the PBGD‐(‐ 112 to + 78) promoter in non‐erythroid cells. We show that, if trans ‐activation is abolished by mutations that destroy either the ‐ 100 CCACC binding or the ‐ 70 GATA binding sites, replacement of the ‐ 100 CCACC binding site by a simian‐virus‐40‐protein‐1 (Sp1) binding site maintains both the erythroid‐specific activity of this promoter and the human GATA‐1 trans ‐activation. Thus, human GATA‐1 acts on the PBGD promoter in association with Sp1 or CCACC binding proteins. This PBGD‐(‐ 112 to + 78) promoter is activated 20‐fold by a cis ‐linked 5′ hypersensitive site 2 (5′HS‐2) of the human β‐globin locus control region. This activation depends on the ‐ 70 GATA and ‐ 100 CCACC or Sp1 binding sites. When a longer ‐ 714 to + 78 fragment of the PBGD promoter is used, the ‐ 70 GATA mutant still displays erythroid‐specific activity and is cis ‐activated by the 5′HS‐2 enhancer, while the ‐ 100 CCACC mutant is completely inactive in the absence or in the presence of the 5′HS‐2 enhancer. Thus, the ‐ 100 CCACC binding site is indispensable for the correct activity and sensitivity of the human PBGD promoter to the 5′HS‐2 enhancer, whereas the ‐ 70 GATA binding site can functionally be replaced by upstream cis ‐acting elements.