Methoxyacetic acid (MAA), a teratogenic toxin, is the major metabolite of ethylene glycol monomethyl ether (EGME, also referred to as 2-methoxyethanol, 2-ME). MAA causes a wide range of toxic effects in laboratory animals including reproductive and developmental toxicity, as well as hematotoxicity, by mechanisms that are not clear. In this study, we employed electron paramagnetic resonance (EPR) spin-labeling techniques in conjunction with spin labels specific for cytoskeletal proteins, bilayer lipids, cell-surface sialic acid, or cell-surface galactose and N-acetylgalactosamine residues of human erythrocyte membranes in order to gain insight into the mechanism of MAA toxicity. The major findings are: (1) MAA significantly increases the protein-protein interactions of skeletal proteins in a concentration-dependent manner (P < 0.001), while 2-ME has no effect (at even a 2.5-fold higher concentration). (2) Addition of MAA leads to significant increase in the rotational motion of spin-labeled terminal galactose and N-acetylgalactosamine residues (2.0 mM MAA, 38% decrease of the apparent rotational correlation time tau a, P < 0.01). (3) The rotational motion of spin-labeled sialic acid, 70% of which is on the major transmembrane sialoglycoprotein (glycophorin A or PAS 1), was not affected by MAA treatment. (4) MAA has no effect on the lipid bilayer fluidity, since no change in the motion of a lipid bilayer specific spin label (5-NS) in the erythrocyte membrane was observed. These results suggest that MAA may lead to teratologic toxicity by interacting not with lipid components but with certain, perhaps specific, protein components, i.e., transport proteins, cytoskeleton proteins or neurotransmitter receptors.