The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)2 D3-treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)2 D3-treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen alpha 1 chain by 1,25-(OH)2 D3 treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)2 D3 treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)2 D3 treatment was also found to induce the alpha subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B-resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)2 D3-treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis.