A soluble enzyme system, isolated from livers of biotin-deficient rats, catalyzes the ATP-dependent synthesis of propionyl holocarboxylase from d-biotin and propionyl apocarboxylase. This system has been resolved by alumina C_∂ gel fractionation into two essential components; (a) gel supernatant which contains propionyl apocarboxylase and (b) gel eluate which contains an enzyme which catalyzes the covalent bonding of d-biotin to propionyl apocarboxylase. The propionyl holocarboxylase synthesis catalyzed by these enzyme systems is irreversible and d·biotin specific. The gel supernatant has been further purified by hydroxyapatite chromatography and (NH₄)₂SO₄ fractionation and the gel eluate by (NH₄)₂SO₄ precipitation and cellulose-phosphate chromatography. An enzyme similar to the gel eluate enzyme has been isolated from cell-free extracts of Propionibacterium shermanii. Although cell-free extracts of P. shermanii do not contain propionyl apo- or holocarboxylase, they do catalyze ATP-dependent propionyl holocarboxylase formation from d·biotin and rat liver propionyl apocarboxylase. Biotin-2'-C¹⁴-labelled propionyl holocarboxylaae, synthesized with these enzyme systems, does not transfer C¹⁴O₂ to propionyl-CoA indicating that the ureido carbon of enzyme-bound biotin is not the"active carbon" of biotin.