The ability of glyceraldehyde-3-phosphate dehydrogenase (GAPD) to associate with 3-phosphoglycerate kinase (3-PGK) in human erythrocytes has been studied. It was found that a stable GAPD-3-PGK complex can be isolated from human erythrocyte hemolysates using immobilized monoclonal antibodies that are specific for GAPD. The complex does not dissociate at high ionic strength (up to 0.3 M NaCl) but is decomposed in the presence of specific ligands interacting with GAPD and 3-PGK, e.g., 1,3-diphosphoglycerate. The interaction between GAPD and 3-PGK isolated from human erythrocytes was investigated. To assess the binding parameters, immobilized GAPD and soluble 3-PGK from erythrocytes were used. About 2.3 moles of monomeric 3-PGK (Kd = 2.4 microM) were bound per mole of the immobilized tetramer of GAPD. Under these conditions the rabbit muscle enzymes form more weak (Kd = 3.8 microM), whereas the yeast enzyme--more stable complexes (Kd = 1.5 microM). No such complexes were detected when the enzyme pairs were isolated from phylogenetically distant sources, such as yeast and mammalian tissues. The species specificity of binding of the two enzymes and possible causes of formation of such stable complexes in erythrocyte lysate are discussed.