The 47,000-D collagen-binding glycoprotein, heat shock protein 47 (HSP47), is a stress-inducible protein localized in the ER of collagen-secreting cells. The location and collagen-binding activity of this protein led to speculation that HSP47 might participate in collagen processing. Chemical crosslinking studies were used to test this hypothesis both before and after the perturbation of procollagen processing. The association of procollagen with HSP47 was demonstrated using cleavable bifunctional crosslinking reagents. HSP47 and procollagen were shown to be coprecipitated by the treatment of intact cells with anti-HSP47 or with anticollagen antibodies. Furthermore, several proteins residing in the ER were noted to be crosslinked to and coprecipitated with HSP47, suggesting that these ER-resident proteins may form a large complex in the ER. When cells were heat shocked, or when stable triple-helix formation was inhibited by treatment with alpha,alpha'-dipyridyl, coprecipitation of procollagen with HSP47 was increased. This increase was due to the inhibition of procollagen secretion and to the accumulation of procollagen in the ER. Pulse label and chase experiments revealed that coprecipitated procollagen was detectable as long as procollagen was present in the endoplasmic reticulum of alpha,alpha'-dipyridyl-treated cells. Under normal growth conditions, coprecipitated procollagen was observed to decrease after a chase period of 10-15 min, whereas total procollagen decreased only after 20-25 min. In addition, the intracellular association between HSP47 and procollagen was shown to be disrupted by a change in physiological pH, suggesting that the dissociation of procollagen from HSP47 is pH dependent. These findings support a specific role for HSP47 in the intracellular processing of procollagen, and provide evidence of a new category of "molecular chaperones" in terms of its substrate specificity and the dissociation mechanism.