The collagen prolyl 4-hydroxylases (P4Hs), enzymes residing within the endoplasmic reticulum, have a central role in the biosynthesis of collagens. In addition, cytoplasmic P4Hs play a critical role in the regulation of the hypoxia-inducible transcription factor HIFalpha. Collagen and HIF P4Hs constitute enzyme families as several isoenzymes have been identified. Two catalytic alpha subunit isoforms have been cloned and characterized for collagen P4Hs from vertebrates, both of them assembling into alpha(2)beta(2) P4H tetramers in which protein disulfide isomerase (PDI) acts as the beta subunit. The catalytic properties of the two isoenzymes are very similar, but distinct differences are found in the binding properties of peptide substrates and inhibitors, and major differences are seen in the expression patterns of the isoenzymes. The nematode Caenorhabditis elegans has five P4H alpha subunit isoforms, PHY1-PHY5. The C. elegans PHY1 and PHY2, together with PDI, are expressed in the collagen synthesizing hypodermal cells and three P4H forms are assembled from them, a PHY-1/PHY-2/PDI(2) mixed tetramer and PHY-1/PDI and PHY-2/PDI dimers. The mixed tetramer is the main P4H form in wild-type C. elegans. PHY-3 is much shorter than PHY-1 and PHY-2, has a unique expression pattern, and is most likely involved in the synthesis of collagens in early embryos. The genome of Drosophila melanogaster contains approximately 20 P4H alpha subunit-related genes, and that of Arabidopsis thaliana six. One A. thaliana P4H has been cloned and shown to be a soluble monomer with several unexpected properties. It effectively hydroxylates poly(L-proline), (Pro-Pro-Gly)(10) and many other proline-containing peptides.