Abstract Diabetes mellitus is associated with a generalized defect in connective tissue metabolism, including decreased growth, poor wound healing, and osteopenia. To determine the role of circulating factors in the etiology of these defects, we studied the effects of diabetic rat serum (DRS) on collagen, the major protein component of connective tissues. After preincubation of costal cartilage from hypophysectomized rats with experimental serum for 20 hours, [ 3H] proline was added for final four hours of incubation. Collagen and noncollagen protein were quantitated using purified bacterial collagenase. Compared to incubation of tissue in buffer without added serum, collagen production in cartilage incubated with 2% DRS was decreased by 23% ( P < .05), and with 4% serum by 88% ( P < .01). In contrast, serum from normal rats (NRS) increased collagen to 158% above buffer-incubated cartilage at 1.0% ( P < .02) and to 196% at 2% serum ( P < .01). Noncollagen protein production decreased below buffer only after addition of 2% or more DRS and increased above buffer after addition of 2% or more of NRS (178%, P < .05). Addition of insulin at 10 and 100 mU/mL to DRS did not reverse defective collagen production, and addition of glucose (up to 900 mg/dL) or ketones (20 mmol/L) to NRS did not induce the changes in collagen production seen after addition of diabetic serum. Chromatographic separation of serum revealed that the inhibitory activity of DRS was in the high molecular weight fraction (>5000). These studies demonstrate that there is a high molecular weight factor(s) in diabetic rat serum that inhibits collagen greater than noncollagen protein production in tissue from nondiabetic animals, and thus may contribute to long-term complications in diabetes through induction of altered connective tissue metabolism.