We have shown previously that expression of the murine CTP:phosphocholine cytidylyltransferase (CT) alpha gene is regulated during cell proliferation (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692). We have now characterized the role of Ha-Ras in the transcriptional regulation of the CTalpha gene. The expression of CTalpha and CTbeta2 proteins and mRNAs was stimulated in C3H10T1/2 murine fibroblasts expressing oncogenic Ha-Ras. Incubation of cells with the specific inhibitor (PD98059) of p42/44(MAPK) decreased the expression of both CT isoforms. Transfection of fibroblasts with CTalpha promoter-luciferase constructs resulted in an approximately 2-fold enhanced luciferase expression in Ha-Ras-transformed, compared with nontransformed, fibroblasts. Electromobility shift assays indicated enhanced binding of the Sp3 transcription factor to the CTalpha promoter in Ha-Ras-transformed cells. Expression of several forms of Sp3 was increased in nuclear extracts of Ha-Ras-transformed fibroblasts compared with nontransformed cells. Tyrosine phosphorylation of one Sp3 form was decreased, whereas phosphorylation of two other forms of Sp3 was increased in nuclear extracts of Ha-Ras-transformed cells. When control fibroblasts were transfected with a Sp3-expressing plasmid, an enhanced expression of CTalpha and CTbeta was observed. However, the expression of CTalpha or CTbeta was not increased in Ha-Ras-transformed cells transfected with a Sp3 plasmid presumably because expression was already maximally enhanced. The results suggest that Sp3 is a downstream effector of a Ras/p42/44(MAPK) signaling pathway which increases CTalpha gene transcription.