Prolyl hydroxylase was purified from extracts of 13-day old chick embryos by an improved affinity column technique. Prolyl hydroxylase was released from the affinity column by poly-L-proline and was separated from the other proteins and from the poly-L-proline by ion exchange chromatography. This improvement allowed eluates from up to six affinity columns to be pooled and chromatographed in a single step. Furthermore, a major contaminating protein which was difficult to remove from the eluted enzyme using the previous procedure was easily separated by the ion exchange column. The overall advantage of the new technique allows much larger amounts of prolyl hydroxylase to be prepared at a single time than was previously possible.