CD19 is a functional component of the B-cell receptor complex where it acts as a modulator of the cellular response to surface immunoglobulin signaling. The gene is expressed from early B-cell developmental stages until the mature B-cell stage, but not in the plasma cell. The human CD19 promoter has been suggested to be regulated by the B-cell specific activator protein, BSAP, the Erg transcription factor and unidentified factors interacting with a GC rich binding site denoted PyG. In this report we present data suggesting that one of the PyG interacting factors is related to mouse early B-cell factor (EBF). Recombinant mouse EBF binds to the PyG site with an affinity about 3-fold lower than to the EBF binding site from the mouse mb-1 promoter in electrophoretic mobility shift assays. Furthermore, the PyG box binds to a factor in nuclear extracts from human B-cell lines, that also interact with the mouse mb-1 promoter EBF binding site. Mutation of the PyG box impaired binding of the factor and the function of a minimal CD19 promoter in human cells of the B lineage, but not in Jurkat T or non-lymphoid HeLa cells. In addition to this, murine EBF was able to activate a wild type but not a PyG mutant human CD19 promoter 7-fold upon transient co-transfection in HeLa cells. Thus, we suggest that a human homologue of mouse EBF participate in transcriptional regulation of the human CD19 promoter.