Bone cells isolated from rat calvaria were cultured on a flat surface, and collagen synthesis was determined by the incorporation of radioactive proline into peptide hydroxyproline. Addition of ascorbic acid to the incubation medium markedly increased the radioactivity of hydroxyproline in cold water-soluble and -insoluble protein fractions, without increasing the radioactivity of peptide-bound proline or the DNA content of the cell cultures. Stimulation was demonstrated with low concentrations of ascorbic acid (3 μM) and was directly proportional to the starting concentration. Hydroxylation of radioactive proline that had previously been incorporated into cell protein was stimulated despite puromycin- or cycloheximide-induced inhibition of protein synthesis. These results indicate that ascorbic acid promotes collagen synthesis in isolated bone cells by directly stimulating the hydroxylation of a proline-rich peptide. Inhibitors of protein synthesis consistently increased the radioactivity of peptide hydroxyproline when added to pulse-labeled cultures in the absence of ascorbic acid. The data suggest that they prevented the dilution of radioactive, unhydroxylated collagen precursor with non-radioactive precursor, thus providing substrate of higher specific activity for hydroxylation.