The transcription factor C/EBPa regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBPa gene do not express detectable levels of the G-CSF receptor and produce no neutrophils. We have recently shown that C/EBPa function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBPa is regulated both in myelopoiesis and leukemogenesis, is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBPa to transactivate the G-CSF receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBPa via the transactivation domain, because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBPa transactivation domain, and does not change C/EBPa DNA binding. Ras acts on serine 248 of the C/EBPa transactivation domain, as it does not enhance the transactivation function of a C/EBPa serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBPa is a PKC consensus site, and a PKC inhibitor blocks the activation of C/EBPa by Ras. Ras signaling phosphorylates C/EBPa on serine 248 in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBPa to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBPa to induce granulocytic differentiation by phosphorylation of serine 248.