Procollagen I is a trimer consisting of two proα1(I) chains and one proα 2(I) chain. In certain cases of mild osteogenesis imperfecta, abnormal proα1(I) chains are degraded very soon after synthesis. As a consequence, the cells produce excess proα2(I) chains, which cannot form trimers and are not secreted. The objective of this work was to determine the intracellular fate of unassociated proα2(I) chains. Mov13 mouse fibroblasts, which do not synthesize proα1(I) mRNA, but do produce proα2(I) mRNA, were incubated with radioactive amino acids using pulse-chase protocols, and proteins were analyzed by gel electrophoresis, autoradiography, and Western blotting. Mov13 cells produced proα2(I) chains that were degraded intracellularly within 30 min. Degradation was inhibited when cells were treated with brefeldin-A, which blocks transit from endoplasmic reticulum to Golgi. Fixed cells exposed to various immunofluorescence markers and imaged by confocal laser scanning microscopy showed that proα2(I) chains colocalized with Golgi and lysosome markers. Degradation was inhibited and chains were secreted when cells were treated with wortmannin, which blocks trafficking to lysosomes. These results demonstrate that unassociated proα2(I) chains leave the endoplasmic reticulum, transit the Golgi, and enter lysosomes where they are degraded.