We have previously identified the functional regions involved in the regulation of human transferrin (Tf) gene expression in the liver and in Sertoli cells of the testis. Here, we show that a different cellular distribution of transcription factors, interacting with the same proximal promoter regions (PRI and PRII), modulates cell type-specific transcription. In the liver, hepatocyte nuclear factor 4 (HNF-4) and the chicken ovalbumin upstream promoter transcription factor (COUP-TF) act at the PRI site, while CCAAT/enhancer-binding proteins (C/EBPs) act at the PRII site. In the testis, distinct combinations of Sertoli proteins SP-A and SP-D and COUP-TF bind to the PRI site, while SP-alpha and SP-beta bind to the PRII site. Cotransfection experiments in Hep3B cells revealed that mostly HNF-4, C/EBP-alpha, C/EBP-delta, and, to a lesser extent, COUP-TF stimulated transcription driven by the -125/+39 region. In Sertoli cells, HNF-4 and COUP-TF appeared to repress, while the C/EBP factors were able to stimulate transcription driven by the -100/+39 region. However, the specific activating combination remains to be defined among the Sertoli proteins. In the non-Tf-expressing HeLa cells, the Tf promoter could be activated by C/EBP-delta. Our data revealed functional antagonism between HNF-4 and COUP-TF, binding to PRI, as well as cross-coupling interactions between HNF-4 and C/EBP, binding to adjacent sites. Thus, cell type-specific DNA-protein interactions, together with protein-protein interactions, may explain the transcriptional regulation of the Tf gene in different cell types.