Short chain acyl-CoA (SCA), medium chain acyl-CoA (MCA), and isovaleryl-CoA (IV) dehydrogenases were purified to homogeneity from human liver using ammonium sulfate fractionation followed by DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5 column chromatographies. The specific activities of the final preparations were enriched 507-, 750-, and 588-fold over those from the second ammonium sulfate fractionation step. The native molecular weights were estimated to be 168,000, 178,000, and 172,000, respectively, by gel filtration. Each of them exhibited, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band with molecular weights of 41,000, 44,000, and 42,000, respectively, indicating a homotetrameric structure. UV/visual spectra, fluorescence spectra, and other evidence indicated that each contains 1 mol of FAD per subunit. They all utilized electron transfer flavoprotein (ETF) or phenazine methosulfate (PMS) as an electron acceptor. The products of SCA dehydrogenase/butyryl-CoA, MCA dehydrogenase/octanoyl-CoA, and IV dehydrogenase/isovaleryl-CoA reactions were identified as crotonyl-CoA, 2-octenoyl-CoA, and 3-methylcrotonyl-CoA, respectively, using gas chromatography. Kinetic parameters Vappmax and Kappm) of these enzymes for various acyl-CoA substrates, as well as Kappm values for ETF and PMS are presented. In general, the substrate specificities of human SCA, MCA, and IV dehydrogenases are slightly less stringent than those of their rat counterparts and resemble those of their bovine and porcine counterparts. The pattern of substrate specificity for these enzymes determined using ETF as electron acceptor significantly differed from that determined using PMS. All of them were severely inhibited by (methylenecyclopropyl)acetyl-CoA.