Cell death was due to activation of the receptor responsive to N-methyl-D-aspartate (NMDA). Even during a 30-min incubation, glutamate toxicity was unaffected by 5 microM CNQX and was greater than 90% blocked by 160 nM MK-801, a specific antagonist of the NMDA receptor. The extent of toxicity was dependent on the age of the cultured cells, the concentration of KCl, which determined membrane polarization, the duration of exposure to, and the concentration of, glutamate. Toxicity was critically pH-dependent, increasing from a minimal effect at pH 7.0 to an extremely high level at pH 8.0. Among a number of acidic amino acids tested, glutamate showed the highest potency. Aspartate, cysteine sulfinate and homocysteate reached the same maximal toxicity, but at a 25 times higher concentration. Cysteate and quinolinate were of very low potency. Zn2+, at 0.5 microM, attenuated glutamate toxicity considerably. At concentrations above 5 microM the metal ion showed strong toxicity on its own. Dialysed serum and serum albumin strongly potentiated glutamate and NMDA toxicity in presence and absence of Mg2+. A concentration of 2 mg/ml bovine serum albumin caused maximal potentiation; fatty acid-free bovine serum albumin and human serum albumin were all about equipotent. To be effective, the serum albumin had to act simultaneously with glutamate. Among a number of proteins and other polymers, only casein showed an ability to potentiate glutamate toxicity, similar to serum albumin. It is concluded that minor effects of glutamate toxicity could be considerably aggravated by the potentiating activity of serum albumin, also in pathological situations in vivo.