AbstractFrequencies of allozyme genotypes are being used as population‐level indicators of environmental heavy‐metal contamination. A genotype of glucose phosphate isomerase,Gpi‐238/38, of mosquitofish (Gambusia holbrooki) has been identified as “mercury‐sensitive” in an acute toxicity assay. Partially purified preparations of GPI‐2 38/38 and GPI‐2 100/100 were assayed to determine differences in maximum gluconeogenic reaction velocity at seven mercury (added as HgCl2) concentrations, 15 to 960 nM Hg. Log‐Probit analysis of the inhibition curves indicated that the log(IC50) (log10of the Hg concentration causing a 50% reduction in reaction velocity) for GPI‐2 100/100 was significantly lower than that for GPI‐2 38/38, even though GPI‐2 100/100 initial uninhibited reaction velocity was greater than that of GPI‐2 38/38. Although the mechanism of inhibition was not experimentally determined, under the assumption of noncompetitive interaction between Hg and GPI‐2, the inhibitor dissociation constants (95% asymptotic C.I.) for GPI‐2 100/100 and GPI‐2 38/38 were estimated from the log (IC50) as 204 nM Hg (155‐269 nM Hg) and 479 nM Hg (363‐617 nM Hg), respectively. These results suggested that Hg susceptibility related to theGpi‐238/38genotype in acute toxicity assays was likely not due to