The subcellular location of hexose diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in baker's yeast (Saccharomyces cerevisiae) was investigated by density gradient centrifugation of spheroplast lysates obtained by osmotic shock treatment of spheroplasts and centrifugation for 10000 g x min. On the evidence obtained from zonal separations these three enzymes of gluconeogenesis are most probably located in the soluble cytosol.