Previous studies suggested that decreased type I collagen synthesis in calvaria of ascorbate-deficient guinea pigs was correlated with weight loss rather than defective proline hydroxylation. The generality of this correlation was examined in articular cartilage, which synthesizes mainly type II collagen, by measuring collagen synthesis and proline hydroxylation in vitro in tissue from ascorbate-supplemented and scorbutic guinea pigs. Ascorbate concentrations in tissues were almost completely depleted after 1 week of deficiency, but proline hydroxylation remained normal until after approximately 3 weeks, when it had decreased only by 10%. At that point collagen synthesis had decreased to about 50% of the control value. There was little additional effect on proline hydroxylation but collagen synthesis decreased further to 20% of normal. Procollagen mRNA levels in cartilage, as measured by dot-blot hybridization with a type II-specific cDNA probe, were unchanged after 2 weeks of scurvy, which correlated with the lack of effect on collagen synthesis during that period. Thereafter, during the period when collagen synthesis decreased, procollagen mRNA levels decreased to 20% of control values. Refeeding ascorbate to acutely scorbutic animals led to reversal of defective proline hydroxylation within 24 h with a slower increase in collagen synthesis and mRNA levels. Collagen synthesis returned to the normal level after 4 days with no further increase, while mRNA levels continued to increase to 2.7 times the control values after 7 days. Thus the major mechanism for regulation of collagen synthesis in articular cartilage during scurvy and ascorbate repletion occurs independently of the effect on proline hydroxylation and is associated with changes in mRNA levels. The lack of precise coordination between collagen synthesis and mRNA levels during repletion, however, suggests that there may be additional regulation through post-transcriptional mechanisms.