Previous studies in this laboratory have identified an essential AP-1 recognition sequence (C1 region; -69 to -63) in th human Pi class glutathione s-transferase (GSTP1) promoter and a negatively acting regulatory element (-105 to -86) that acts to suppress GSTP1 transcription in the human mammary carcinoma cell line, MCF7 (1). The data presented here further delineate the functional characteristics of the GSTP1 promoter by examining the significance of two potential binding sites for the transcription factor, Sp1 (-57 to -49 and -47 to -39). The introduction of mutations within these Sp1-like elements and the use of Sp1 antisera in electrophoretic mobility shift assays demonstrated that Sp1 was bound to this region of the GSTP1 promoter in three different cell lines, MCF7, VCREMS, and EJ. Moreover, these in vitro studies indicated that only one of the two putative Sp1 response elements was utilized. Transient transfection assays using GSTP1 promoter constructs that incorporated mutations of the Sp1 elements clearly demonstrated that binding of Sp1 to the GSTP1 promoter was absolutely required for optimal levels of GSTP1 transcription. In particular, disruption of the distal Sp1 recognition motif (-57 to -49) markedly reduced GSTP1 promoter activity in each cell line, thus indicating preferential binding of Sp1 to the distal site. However, insertion of the repressor binding site (-105 to -86) into these constructs suggested that Sp1 was not involved in mediating the suppressive effects of the GSTP1 transcriptional repressor in MCF7 cells, because inhibition of Sp1 binding did not alleviate repressor activity. Therefore, these studies provide strong evidence that Sp1 plays a central role in regulating basal levels of GSTP1 transcription.