The intracellular location of pyruvate carboxylase (EC 6.4.1.1) in rat liver and Saccharomyces cerevisiae was investigated using the antibody-gold and protein A-gold techniques carried out as a postembedding immunoelectron microscopic procedure. The vast majority of gold particles (greater than 98%), indicative of the presence of antigenic sites of pyruvate carboxylase, were found in the mitochondria of rat liver. No other cellular compartment was labeled except the cytosol which did not account for more than 2% of the total labeling of a rat hepatocyte. Furthermore, 60% of labeled pyruvate carboxylase molecules within a mitochondrion were found adjacent to the matrix side of the inner mitochondrial membrane. In contrast, in S. cerevisiae, pyruvate carboxylase was found exclusively in the cytosol.