We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated “A gamma-neo” transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.