Ribonucleic acid extracted from chick embryo calvaria directs the synthesis of a collagenous product in a cell-free system. A portion of the prolyl residues incorporated into this protein can be hydroxylated in vitro by prolyl hydroxylase as demonstrated by the release of 3H-H20 from 3H-proline and by the direct demonstration of 3H-hydroxyproline in acid hydrolysates of the product. Thirty percent of potential sites become hydroxylated in the in vitro reaction using either cell-free product or an underhydroxylated collagen extracted from chick calvaria. Neither prolonged incubation nor increased levels of enzyme increased the extent of hydroxylation in either substrate.