An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum-free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10(-6) M) and transferrin (5 micrograms/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10(-4) M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism.