We have shown previously that hydroxylation played a critical role in the trimer assembly and disulfide bonding of the three constituent alpha chains of a minicollagen composed of the extreme C-terminal collagenous (COL1) and noncollagenous (NC1) domains of type XII collagen in HeLa cells (Mazzorana, M., Gruffat, H., Sergeant, A., and van der Rest, M. (1993) J. Biol. Chem. 268, 3029-3032). We have further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric disulfide-bonded type XII minicollagen in an insect cell expression system. For this purpose, type XII minicollagen was produced in insect cells from baculovirus vectors, alone or together with wild-type human prolyl 4-hydroxylase or with the human enzyme mutated in the catalytic site of its alpha or beta subunits or with the individual alpha or beta subunits. When type XII minicollagen was produced alone, negligible amounts of disulfide-bonded trimers were found to be produced by the cells. However, coproduction of the collagen with the two subunits of the wild-type human enzyme dramatically increased the amount of disulfide-bonded trimeric type XII minicollagen molecules. In contrast, coproduction of the collagen with alpha subunits that had a mutation completely inactivating the human enzyme failed to enhance the trimer assembly. These results directly show that an active prolyl 4-hydroxylase is required for the assembly of disulfide-bonded trimers of type XII minicollagen.