Chick cranial-bone procollagen, extracted at neutral pH in the presence of inhibitors of proteolytic enzymes, exists as a triple-stranded protein with disulfide bonds linking all three chains. The biosynthetic precursor function of this procollagen was demonstrated by pulsechase experiments. The ratio of radioactive hydroxyproline to proline in proalpha chains obtained by reduction and alkylation of the disulfide-bonded precursor was similar to the value determined for proalpha1 from acid-extracted procollagen. However, the molecular weight of these chains was higher than that previously determined for proalpha1 and proalpha2, suggesting that extraction of tissue at low pH results in a selective loss of disulfide-bonded regions from procollagen. These findings reconcile several apparently conflicting reports on the nature of procollagen identified in bone and in the medium of cultured fibroblasts and indicate that in both systems procollagen is synthesized as a disulfidelinked protein.