Deficiency of propionyl-CoA carboxylase (PCC) results in propionic acidemia, an autosomal recessive disorder characterized by ketoacidosis sufficiently severe to cause neonatal death. PCC is involved in the catabolism of branched-chain amino acids, odd-chain fatty acids, and cholesterol. The enzyme is a biotin-dependent mitochondrial protein composed of two heterologous subunits arranged into an 800-kDa a 6 b 6 dodecameric structure. Approximately 60 mutations have been reported in the nuclear genes PCCA and PCCB that encode the two PCC subunits. The vast majority of these mutations have not been examined at the protein level. We present an initial characterization of 13 mutations located in exons 1, 37, and 1214 of PCCB. After expression in E. coli, these recombinant mutant enzymes were analyzed for stability, biotinylation, ab subunit interaction, and activity. Our results show a functional dichotomy in these PCCB mutations with some mutants (R44P, S106R, G131R, G198D, V205D, I408del, and M442T) capable of varying degrees of assembly but forming catalytically inactive PCC proteins. Other PCCB mutants (R165W, E168K, D178H, P228L, and R410W) that are PCC deficient in patient-derived fibroblasts, were found to be capable of expressing wild-type level PCC activity when assembled in our chaperoneassisted E. coli expression system. This result indicates that these mutations exert their pathogenic effect due to an inability to assemble correctly in patients cells. This initial screen has identified a range of mutant PCC proteins that are sufficiently stable to be purified and subsequently used for structurefunction analysis to further elucidate the complex relationship between genotype and phenotype in propionic acidemia. Hum Mutat 19:629640, 2002.