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Phosphatidylcholine (PtdCho) biosynthesis was examined in the sterol regulatory defective (SRD) Chinese hamster ovary (CHO) cell line SRD 6. SRD 6 cells do not display transcriptional activation of sterol-regulated genes and are cholesterol auxotrophs. Compared to CHO 7 cells (parental line from which the SRD cells were derived), incorporation of [3H]choline during a 2-h pulse into PtdCho and sphingomyelin was reduced 3- and 4.5-fold, respectively, in SRD 6 cells grown with or without cholesterol. SRD 6 cells grown in cholesterol-free medium for 24 h had 8% less phosphatidylcholine (PtdCho) mass compared to CHO 7 cells. Consistent with impaired CTP:phosphocholine cytidylyltransferase activity, [3H]choline-labeled SRD 6 cells had elevated [3H]phosphocholine and delayed conversion to [3H]PtdCho during a 2-h chase period. Compared to CHO 7 cells, cytosolic cytidylyltransferase activity was elevated 2- to 3-fold in SRD 6 cells grown in the absence of cholesterol, but activity in the total membrane fraction was normal. Immunoblot analysis confirmed that cytidylyltransferase mass was increased 2-fold in SRD 6 total cell extracts and cytosol, but not membranes. The amount of [32P]phosphate-labeled cytidylyltransferase in cytosol and membranes of SRD 6 cells were similar to controls. Likewise, cytidylyltransferase mRNA levels were not significantly different between SRD 6 and CHO 7. The defect in PtdCho synthesis in SRD 6 cells could be overcome by treatment with 150 microns oleate, such that after 5 h [3H] choline incorporation into PtdCho and phosphocholine in SRD 6 and CHO 7 cells was similar. Cholesterol auxotrophic SRD 6 cells display reduced PtdCho mass and synthesis and elevated levels of cytosolic cytidylyltransferase, defects that were only partially corrected by growth in exogenous cholesterol. These results indicate a requirement for normal cholesterol regulation and synthesis in the maintenance PtdCho levels and activity of cytidylyltransferase.