The results presented here show that isolated subunits of transcarboxylase specifically catalyze the two partial reactions of transcarboxylation as shown in eq 1-3. The 12S central subunit is active in the transcarboxylation with methylmalonyl-CoA but inactive with oxalacetate and the peripheral metallo 5S subunit is active in the transcarboxylation with oxalacetate but inactive with methylmalonyl-CoA. These subunits, likewise, are specific for the reverse partial reactions; the central subunit catalyzing transfer from the carboxylated biotinyl group to propionyl-CoA to yield methylmalonyl-CoA and the peripheral subunit to pyruvate to yield oxalacetate. Thus, the central subunit contains the sites for the CoA esters (methylmalonyl-CoA and propionyl-CoA) and the peripheral metallo subunits for the keto acids (oxalacetate and pyruvate). In the overall reaction the biotinyl carboxyl carrier protein acts as a shuttle to carry the carboxyl groups between the two subunits. Biotin and certain biotin analogs are inactive in these partial reactions but the similar to 40- or similar to 66-residue biotinyl peptides, which are derived from the carboxyl carrier protein, are active. Transcarboxylase can be reconstituted from its isolated subunits and a comparison was made of the rate of the overall reaction when the subunits were assembled, as in the intact enzyme, with that obtained when the reaction was catalyzed by the nonassembled subunits. In the latter case, since the biotinyl carboxyl carrier subunit must diffuse from one subunit to the other, the overall reaction is much slower than with the assembled subunits. The reaction with trypsinized transcarboxylase from which the similar to 66-residue and similar to 40-residue biotinyl peptides have been stripped, likewise, was slow even though the biotinyl peptides were added to the reconstitution mixture. The 12SH and 5SE subunits remain assembled after trypsin treatment but the biotinyl peptides apparently do not combine firmly or properly with the trypsinized enzyme and the biotinyl group apparently must oscillate as a carboxyl carrier between the two sites on the subunits by diffusion.