The -198 T-->C and the -175 T-->C transitions involving the proximal gamma-globin gene promoter are associated with the hereditary persistence of fetal hemoglobin (HPFH) phenotype and have been demonstrated to increase promoter activity in erythroid cells using transient and stable transfection systems. The above base changes are thought to alter the binding of different transcription regulatory proteins. Another mutation of the proximal gamma-globin promoter, -158 C-->T, has been less clearly linked to the HPFH phenotype but has been associated with increased G gamma activity. In the present paper, the -198 T-->C, -175 T-->C and -158 C-->T mutations both singly and in various combinations were evaluated by an in vitro expression assay. gamma-Globin promoters were transfected by electroporation into K562 human erythroleukemia cells and their activity measured in a human growth hormone (hGH) reporter gene assay. A novel cotransfectant was used to assess transfection efficiency. Results confirmed the previously reported upregulation of gamma-globin activity with the -198 T-->C and -175 T-->C HPFH mutations and a cooperative effect on promoter activity when both these mutations are present in cis. No effect of the -158 C-->T mutation was seen either alone or in combination with the -175 T-->C and -198 T-->C mutations.