We have cloned and sequenced cDNAs encoding the α-subunit of rat liver propionyl-CoA carboxylase (PCC), a biotin-dependent, mitochondrial matrix protein. The full-length cDNA spans 3327 base pairs, has a long (895 base pairs) 5'-untranslated region, and encodes a protein of 721 amino acids. In vitro transcription and translation of the full-length rat α-PCC cDNA produces a product which is immunoprecipitable with antibodies specific to PCC and has the same apparent molecular weight as in vivo synthesized α-PCC. Rat liver α-PCC precursor is not quantitatively biotinylated when synthesized in a cell-free rabbit reticulocyte system. Nevertheless, when incubated with isolated rat liver mitochondria in vitro, the precursor is imported and proteolytically cleaved to its mature form. A sequence comparison of rat liver α-PCC and other biotinylated polypeptides reveals the absolute conservation of three glycines and one valine in the region surrounding the biotinylated lysine residue. This pattern of conserved residues is also present in lipoylated proteins, where lipoic acid is similarly, covalently attached to a lysine residue. A possible functional role for the conserved glycine residues and further sequence similarity surrounding the site of biotinylation and lipoylation is discussed.