We have recently demonstrated that both methylmercury (MeHg) and mercuric chloride (MC) induce d-aspartate release from neonatal rat primary astrocyte cultures maintained in isotonic conditions [1,3,21,22]. In the present study, we compare several other sulfhydryl-(-SH) selective alkylating reagents [methyl methanethiosulfonate (MMTS), N-ehtylmaleimide (NEM), and iodoacetamide (IA)] in isotonic, as well as hypotonic conditions to discern the functional importance of -SH groups in [ 3H] d-aspartate and 86rubidium ( 86Rb) release from astrocytes. Treatment of astrocytes (5 min) in isotonic buffer with the hydrophobic reagent NEM (10 μM) caused a marked increase in 86Rb release but had no effect of [ 3H] d-aspartate release. Neither IA-, nor MMTS-treatment (both at 10 μM) induced increase in [ 3H] d-aspartate of 86Rb release in isotonic buffer. In hypotonic condition (−50 mM Na +), astrocytes were most sensitive to MC exposure (5 μM), exhibiting an increase in both [ 3H] d-aspartate and 86Rb efflux. The hydrophobic compounds MMTS and NEM, and the hydrophilic -SH modifying reagent, IA, attenuated the hypotonic-induced efflux of [ 3H] d-aspartate, in the absence of an effect of 86Rb release. These observations are consistent with a critical role for -SH groups both in basal (i.e. isotonic) and hypotonic-induced release of d-aspartate and Rb from astrocytes. Lack of uniformity of these effects may be attributed to site-specificity, related to the physicochemical properties of these -SH alkylating reagents.