The expression of the CTP: phosphoethanolamine cytidylyltransferase gene Pcyt2 is significantly up-regulated during C2C12 muscle cell differentiation, which was demonstrated by elevated Pcyt2 protein ( approximately 2.3-fold), mRNA ( approximately 2.6-fold) and promoter activity ( approximately 2-fold) in myotubes relative to myoblasts. Mutation and 5' deletion analyses of Pcyt2 promoter established the minimal core sequence and three main upstream regulatory regions. The core promoter (-111/+29 bp) strongly depends on the binding of cEBP to an inverse CCAAT-box located at position -82/-77 bp. Transcription factors Sp1 and Sp3 bind to regions A (-508/-378 bp) and C (-157/-111 bp), and muscle-specific differentiation factor MyoD targets the region C. Region B (-228/-157 bp) is weakly regulated by Sp factors and binds unknown protein complexes that acts as negative regulatory elements. Sp1 is less present in myotubes than in myoblasts and when over-expressed in myotubes significantly reduces promoter activity. These results demonstrate that elevated content of MyoD, reduced content of Sp1, and changed ratio of Sp1 to Sp3 all together contributed to a stimulated transcription of Pcyt2 gene during muscle cell differentiation.