Pyridinoline is a non-reducible crosslink of collagen. The changes in the content of this crosslink during in vitro aging of bone collagen were studied. The pyridinoline content of bone collagen of 6-week-old chick was very low. When the collagen was incubated with physiological saline in air at 37 degrees C, the pyridinoline content increased markedly. After 6 weeks, the content reached the same level as that of the adult. The synthesis of pyridinoline was inhibited when air was excluded. The in vitro aged collagen was less susceptible to pepsin than the untreated collagen. The results indicate that pyridinoline is one of the mature crosslinks of collagen and plays a role in the stabilization of collagen fibers.