Diabetics may have normal somatomedins by radioimmunoassay yet decreased somatomedin activity by bioassay. The discrepancy appears due to circulating inhibitory factors; inhibitors in whole diabetic serum antagonize the action of both somatomedins and insulin via noncompetitive interactions. Since little is known about the nature of the inhibitory activity, serum from streptozotocin-diabetic rats was fractionated, and inhibitory activity measured as the ability of fractions to blunt stimulation of SO4 uptake by hypophysectomized rat costal cartilage exposed in vitro to somatomedins in normal rat serum. After establishing that inhibitory activity was stable to pH and lyophilization, diabetic rat serum was gel filtered at pH 7 (somatomedins bound to carrier proteins) and 2.4 (somatomedins dissociated, mol wt approximately 8000). Using Sephadex and Sephacryl columns at neutral pH, inhibitors were detected at mol wt approximately 250,000, approximately 24,000, and approximately 940. Predominant activity was at approximately 24,000 and approximately 940. In contrast, Sephadex columns at acid pH revealed inhibitors only at mol wt approximately 21,000. Diabetic rat serum was also subjected to ion-exchange chromatography on CM-Sepharose. A single band of activity at pH 5-7 was found on elution with increasing pH, suggesting an isoelectric point(s) lower than that of the somatomedins. However, three areas of activity were seen on elution with increasing ionic strength at pH 5-at 0.02 M and 0.14-1.4 M and at 2.0 M pH 5.0-5.5. These studies indicate that reduced anabolism in diabetes may be due in part to three species of circulating somatomedin inhibitors, largely of mol wt approximately 21-24,000 and approximately 940, and also of mol wt approximately 250,000.(ABSTRACT TRUNCATED AT 250 WORDS)