Collagen biosynthesis is a complex process that begins with the association of three procollagen chains. A series of conserved intra- and interchain disulfide bonds in the carboxyl-terminal region of the procollagen chains, or C-propeptide, has been hypothesized to play an important role in the nucleation and alignment of the chains. We tested this hypothesis by analyzing the ability of normal and cysteine-mutated pro-alpha2(I) chains to assemble into type I collagen heterotrimers when expressed in a cell line (D2) that produces only endogenous pro-alpha1(1). Pro-alpha2(I) chains containing single or double cysteine mutations that disrupted individual intra- or interchain disulfide bonds were able to form pepsin resistant type I collagen with pro-alpha1(I), indicating that individual disulfide bonds were not critical for assembly of the pro-alpha2(I) chain with pro-alpha1(I). Pro-alpha2(I) chains containing a triple cysteine mutation that disrupted both intrachain disulfide bonds were not able to form pepsin resistant type I collagen with pro-alpha1(I). Therefore, disruption of both pro-alpha2(I) intrachain disulfide bonds prevented the production and secretion of type I collagen heterotrimers. Although none of the individual disulfide bonds is essential for assembly of the procollagen chains, the presence of at least one intrachain disulfide bond may be necessary as a structural requirement for chain association or to stabilize the protein to prevent intracellular degradation.