Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their alpha subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19-20- and 35-residue peptides corresponding to the two sites in HIF-alphas and purified recombinant HIF-1alpha and HIF-2alpha ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the K(m) values to 90-800 nm, i.e. to 0.7-11% of those for the shorter peptides, whereas those representing the N-terminal site were 10-470 microm, i.e. 10-135%. The K(m) values of HIF-P4H-1 for the recombinant HIF-alpha ODDDs were 10-20 nm, whereas those of HIF-P4H-2 and -3 were 60-140 nm, identical values being found for the wild-type HIF-1alpha ODDD and its N site mutant. The K(m) values for the C site mutant were about 5-10 times higher but only 0.2-3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF-P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The K(m) values of HIF-P4H-2 for oxygen determined with the HIF-1alpha ODDD and both its mutants as substrates were all about 100 microm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O(2) levels, indicating that HIF-P4Hs are effective oxygen sensors.