We have examined factor binding to the distal human gamma-globin CCAAT site and three naturally occurring hereditary persistence of fetal haemoglobin (HPFH) mutations of this site. Factor binding was examined using nuclear extracts from the erythroleukaemic cell lines K562 and MEL, and from A4 cells, a non-transformed mouse bone marrow stem cell line, using the electrophoretic mobility shift assay. Under standard binding conditions, in addition to the previously reported binding by a CCAAT factor (CP1) and GATA-1, the wild-type (wt) sequence bound high mobility factors which appeared to be GATA-2 isoforms. However, when the non-specific competitor conditions were varied, the binding profile with K562, but not MEL nuclear extract, was substantially altered. CP1 and GATA-1 were absent, and two new factors were detected, one of which bound preferentially to the Greek and Japanese non-deletion HPFH mutants. However, binding by the GATA-2 isoforms to the wt sequence was maintained with both cell types, as it was using the A4 cell line. With modified binding conditions, in A4 cells the two non-deletion and the Black deletion HPFH mutants each had a different protein binding profile which was lost on erythroid induction of the cells. We discuss the possibility that the GATA-2 isoforms bound to the wt sequence may function to suppress wt gamma gene expression in the bone marrow. Additionally, those factors which bind preferentially either to the deletion or non-deletion HPFH mutants may play positive roles in establishing an active chromatin structure.