Abstract Synthetic polytripeptides with the structure (l-Pro-Gly-l-Pro)n were used to study protocollagen hydroxylase, the enzyme which hydroxylates appropriate proline residues in protocollagen, the polypeptide precursor of collagen. With these synthetic substrates the synthesis of hydroxyproline could be followed quantitatively, and for the first time the reaction was studied under conditions where the substrate concentration was not rate-limiting. As previously observed with protocollagen as a substrate for the reaction, the hydroxylation of (Pro-Gly-Pro)n required α-ketoglutarate, ferrous iron, and ascorbate, but the Km values for the cofactors were slightly different from those observed with protocollagen. Hydroxylation of (Pro-Gly-Pro)n fractions of varying molecular weights indicated that a fraction with a molecular weight of about 1,800 had a Km value of 220 µg per ml, and four fractions ranging in molecular weight from 4,000 to 15,000 gave Km values of 110 µg per ml. All the Km values for (Pro-Gly-Pro)n preparations were considerably greater than that previously reported for proline-labeled protocollagen. No differences were observed in the maximal velocities with the different fractions of (Pro-Gly-Pro)n. A copolymer of glycine and proline (1:1.9) with a molecular weight of 4000 gave a Km of 440 µg per ml, and the maximal velocity was less than that observed with (Pro-Gly-Pro)n preparations. Poly-l-proline II was a competitive inhibitor of the enzyme, and the Ki value observed was 0.3 µg per ml. The results suggest that (Pro-Gly-Pro)n is not as appropriate a substrate for the hydroxylase as protocollagen, and that the higher Km values observed for preparations of (Pro-Gly-Pro)n were not explained by their smaller size. The results also suggest that the enzyme has a high affinity for proline in the extended single-stranded helix found in poly-l-proline II.