Chironomus riparius Meigen were exposed to three different insecticides, the organophosphorous fenitrothion and the carbamates carbaryl and carbofuran (0, 1, 10, and 100 microg/L) for 24h as fourth-instar larvae. Acetylcholinesterase (AChE), naphtylacetate esterase (NAE), p-nitrophenylacetate esterase (PNPAE), glutathione-S-transferase (GST), and a number of metabolites (alanine, pyruvate, lactate, trehalose, aspartate, oxalacetate) were measured to determine which was the most valuable biochemical biomarker of exposure. AChE activity was significantly reduced by all three insecticides, PNPAE by fenitrothion, carbofuran and carbaryl, whereas NAE activity was stimulated by carbaryl and unaffected by fenitrothion and carbofuran. Metabolites analysis revealed a strong accumulation of alanine in larvae exposed to fenitrothion, but not in larvae exposed to carbamates. This accumulation was accompanied by a significant increase of lactate and a significant decrease of pyruvate and trehalose. No variations were observed with carbofuran and carbaryl. No change of aspartate concentration was detected. We conclude that the association of alanine accumulation with a significant inhibition of AChE activity can be used as a valuable biochemical biomarker of exposure.