Methylmercury (MeHg) is highly neurotoxic with an apparent dose-related latency period between time of exposure and the appearance of symptoms. Astrocytes are known targets for MeHg toxicity and a site of mercury localization within the central nervous system (CNS). Glutamine synthetase (GS) is an enzyme localized predominately within astrocytes. GS converts two potentially toxic molecules, glutamate and ammonia, to the relatively non-toxic amino acid, glutamine. During prolonged exposure to MeHg, inorganic mercury (I-Hg) accumulates within the brain, suggesting in situ demethylation of MeHg to I-Hg. To determine if speciation of mercurials would differentially alter GS activity and expression, neonatal rat primary astrocyte cultures were exposed to MeHg or mercuric chloride (HgCl2) for 1 or 6 h. MeHg produced no changes in GS activity, protein, or mRNA at any time or dose tested. In contrast, HgCl2 produced a dose dependent decrease in astrocytic GS activity at both 1 and 6 h. There were no changes in GS protein or mRNA levels following HgCl2 exposure. Additional studies were carried out to determine GS activity in cell lysates incubated with HgCl2 or MeHg. In cell lysates, HgCl2 was three-times more potent than MeHg in inhibiting GS activity. The inhibition of GS activity in cell lysates by HgCl2 was reversed by the addition of dithiothreitol (DTT), while DTT did not restore GS activity following MeHg. These data suggest that astrocytic GS activity is not inhibited by physiologically relevant concentrations of MeHg, but is inhibited by I-Hg, which is present in CNS following chronic MeHg exposure.