Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of CTP:phosphocholine cytidylyltransferase alpha (CT) are not fully understood. In this study, we investigated whether or not histone deacetylation is involved in repression of CT expression in quiescent C3H10T1/2 mouse embryo fibroblasts. We have examined the contributions of the Sp1 and E2F binding sites in the repression of CT gene expression. Immunoprecipitation experiments showed that histone deacetylase 1 (HDAC1) and HDAC activity are associated with Sp1 in serum-starved cells or during serum stimulation. However, HDAC1 association with E2F was only detected in serum starved cells. By chromatin immunoprecipitation assays we detected both direct and indirect association of HDAC1 with the CT promoter. Treatment with the HDAC inhibitor Trichostatin A induced CT expression. Our data suggest that HDAC1 plays a critical role in CT repression, and that Sp1 and E2F may serve as key targets for HDAC1-mediated CT repression in fibroblasts.