Propionyl-CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme, composed of an equal number of alpha and beta subunits, that functions in the catabolism of branched-chain amino acids and other metabolites. Mutations of the PCCA (alpha subunit) or PCCB (beta subunit) gene cause the inherited metabolic disease, propionic acidemia. We report the cloning of a full-length cDNA encoding the beta subunit of human PCC. The open reading frame encodes a pre-beta polypeptide of 539 amino acids (58,205 Da). The cDNA was introduced into the expression vector, pRc/CMV, and microinjected into the nucleus or, as ribotranscripts, into the cytoplasm of fibroblast lines from patients with defects of the beta subunit. The restoration of function was monitored by autoradiography of PCC-dependent [14C]-propionate incorporation into cellular protein. These results confirm the completeness of the clone and demonstrate the capacity for beta subunits derived from the microinjected cDNA or RNA to be transported into mitochondria and assembled with endogenously derived alpha subunits to form functional PCC.