The c-myb oncogene encodes a nuclear binding protein which may play a major role in differentiation during early T cell development. However, the functionally important transcription regions in the GC promoter site have not been defined and the significance of the regulation of this promoter site in T cell differentiation has not been determined. Therefore, the promoter strength was determined by measurement of the CAT activity in cell extracts of EL-4 cells that were transfected with a CAT expression vector that contained cloned segments of the 5′ myb gene. Stepwise removal of DNA sequences between -2300bp and -346bp upstream from the ATG initiation codon resulted in a gradual loss of 50% of CAT activity, whereas deletion of DNA sequences from -346 to -295 and -232 to -155bp upstream from the ATG initiation codon eliminated promoter activity. On analysis of the CAT activity after transfection of various cell lines with these same constructs, it was found that the same two promoter regions were required for high CAT activity in all the cell lines, including murine cell lines which express the α /β TCR and high levels of c-myb (BW5147), the α/β TCR and low levels of c-myb (Yac-1), or the γ/δ TCR (KN 12.1 and KN 2.4 T), a murine fibroblast T cell line (NIH-3T3), and a human epithelial cell line (HeLa). However, the CAT activity did not correlate with steady state levels of expression of the c-myb gene in the murine cell lines. Our data indicate that the c-myb oncogene promoter is constitutively expressed, is highly dependent on a limited region of the 5′ myb gene, requires two DNA elements for optimal activity, and is functional in diverse T cell lines. © 1993.