4-Hydroxyproline is found in collagens, collagen-like proteins, elastin, and the hypoxia-inducible transcription factor in animals and in many hydroxyproline-rich glycoproteins in plants. We report here on the cloning and characterization of a second plant P4H (prolyl 4-hydroxylase), At-P4H-2, from Arabidopsis thaliana. It consists of 299 amino acids and shows 33% sequence identity to the first characterized isoenzyme, At-P4H-1. A characteristic feature of the At-P4H-2 polypeptide is a 49-amino-acid C-terminal toxin homology domain with 6 cysteines that is not found in At-P4H-1 but is present in a putative rice P4H homologue. At-P4H-2 differed distinctly from At-P4H-1 in its substrate specificity. Recombinant At-P4H-2 hydroxylated poly(L-proline) and extensin and arabinogalactan-like peptides effectively but with much higher Km values than At-P4H-1, suggesting different roles for the two At-P4Hs in the plant cell. Unlike At-P4H-1, At-P4H-2 hydroxylated collagen-like peptides only very inefficiently and did not hydroxylate hypoxia-inducible transcription factor alpha-like peptides at all. All the peptides efficiently hydroxylated by At-P4H-2 had at least 3 consecutive prolines, suggesting that these may represent a minimum requirement for efficient hydroxylation by this isoenzyme. N-terminal sequencing of an extensin-like peptide SPPPVYKSPPPPVKHYSPPPV indicated that At-P4H-2 preferentially hydroxylated the 3rd proline in the C-terminal PPP triplet. The Km values of At-P4H-2 for the reaction cosubstrates Fe2+, 2-oxoglutarate, and ascorbate were similar to those of At-P4H-1 with the exception that the Km for iron was about 3-fold lower. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate, well known competitive inhibitors of the vertebrate P4Hs with respect to 2-oxoglutarate, were also competitive inhibitors of At-P4H-2 but with Ki values 5-100-fold higher than those of human type I collagen P4H. It thus seems that there are some distinct differences in the structure of the 2-oxoglutarate-binding site between At-P4H-2 and the animal collagen P4Hs.