Oct-4 is expressed in embryonic stem cells and is one of the first transcription factors differentially regulated during mouse development. Recent ablation of Oct-4 function in vivo has demonstrated that its expression is essential for preventing embryonic cell differentiation. Several studies have indicated that activation of the Oct-4 TATA-less promoter is mediated by a GC-box representing a high affinity binding site for the transcription factor Sp1. In this study, we have analyzed Oct-4 expression and the activity of the Oct-4 promoter GC box in Sp1 deficient ES cells. We found that in these cells, Oct-4 RNA and protein levels were comparable to wt ES cells and the activity of the Oct-4 promoter GC-box was even increased. Furthermore, the pattern of Oct-4 down-regulation in Sp1 deficient ES cells was unchanged compared to wt cells. Analysis of GC-box binding proteins in extracts from Sp1-/- ES cells revealed that Sp3, another member of the Sp1 transcription factors family, efficiently bound to Sp1 site. These results show that in ES cells Sp1 is not essential for Oct-4 gene activity. Thus, Sp1 function in undifferentiated embryonic cells may be complemented by Sp3 and/or by other GC-box binding transcription factors.