Progression of MCF-7 cells from early passage (MCF-7E, <200 passage) to late passage (MCF-7L, >500 passage) correlates with a loss of sensitivity to exogenous TGFbeta1. We have previously shown that loss of TGFbeta sensitivity is due to decreased expression of the transforming growth factor receptor type II (TbetaRII) and is associated with increased tumorigenicity in nude mice. Reduced TbetaRII expression in MCF-7L cells is caused by decreased TbetaRII promoter activity in this cell line. Our previous studies using 5' deletion constructs of this promoter revealed that MCF-7L cells were unable to support transcription of the minimal promoter (-47 to +2) to the same levels as the MCF-7E cells. This region of the promoter contains an Sp1 element at position -25 from the major transcription start site. In this study, we investigated the role of Sp1 in TbetaRII transcription. Mutation of the Sp1 site resulted in decreased transcription of TbetaRII in MCF-7E and MCF-7L cells, indicating that this site played a role in transcription of this promoter. Gel shift assays using the proximal Sp1 site from the TbetaRII promoter showed enhanced DNA:protein complex formation with nuclear proteins isolated from MCF-7E cells compared with MCF-7L cells. Supershift analysis identified this binding activity as Sp1. Western blot analysis of Sp1 levels demonstrated that MCF-7E cells contain increased Sp1 protein compared with MCF-7L cells, paralleling the increased binding activity. Differential Sp1 activity was also demonstrated by higher levels of transcription of an Sp1-dependent insulin-like growth factor II promoter construct in MCF-7E cells compared with MCF-7L cells. Co-transfection of an Sp1 expression vector with a TbetaRII promoter construct in MCF-7L cells induced the expression from the promoter-CAT constructs and resulted in an increase of endogenous TbetaRII protein levels. These results demonstrate that the transcriptional repression of TbetaRII in MCF-7L cells is caused, in part, by lower Sp1 levels.