The inherited metabolic disease propionic acidemia (PA) can result from mutations in either of the genes PCCA or PCCB, which encode the α and β subunits, respectively, of the mitochondrial enzyme propionyl CoA-carboxylase. In this work we have analyzed the molecular basis of PCCA gene defects, studying mRNA levels and identifying putative disease causing mutations. A total of 10 different mutations, none predominant, are present in a sample of 24 mutant alleles studied. Five novel mutations are reported here for the first time. A neutral polymorphism and a variant allele present in the general population were also detected. To examine the effect of a point mutation (M348K) involving a highly conserved residue, we have carried out in vitro expression of normal and mutant PCCA cDNA and analyzed the mitochondrial import and stability of the resulting proteins. Both wild-type and mutant proteins were imported into mitochondria and processed into the mature form with similar efficiency, but the mature mutant M348K protein decayed more rapidly than did the wild-type, indicating a reduced stability, which is probably the disease-causing mechanism.