Objective Endosulfan is an extremely toxic organochlorine pesticide to aquatic organisms which might be hampering fish health through impairment of metabolism sometimes leading to death.So an experimental protocol was designed to look at endosulfan effects on a number of selected biochemical endpoints as well as to develop a mechanistic understanding of biochemical effects of endosulfan in freshwater fish. Methods The adult freshwater catfish Clarias batrachus were collected and acclimatized to laboratory condition for two weeks prior to experimentation. The toxicity bioassay test of commercial grade endosulfan (35% EC) was conducted for 21 days to determine its initial lethal concentration. The fish were exposed to sublethal concentration of endosulfan (0.06 mg/L) for 21 days. Pesticide - withdrawal experiments were also performed to study recovery. Protein synthesis inhibitors were injected to know the possible mechanism of recovery. The specimens of C. batrachus were sacrificed and brain, liver and caudal white skeletal muscle were removed. Tissues were homogenized and fractions were obtained by differential centrifugation. The activities of citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH) and lactate dehydrogease (LDH) were assayed spectrophotometrically. Similarly, DNA, RNA and protein content were measured as per standard procedure. Results The exposure of sublethal concentration of endosulfan decreased the activity of citrate synthase (CS) and glucose 6-phosphate dehydrogenase (G6-PDH) in the brain, liver and skeletal muscle of the freshwater catfish, C. batrachus. The brain lactate dehydrogenase (LDH) activity was also reduced in response to endosulfan toxicity. The maximum reduction in activities of these enzyme was 34%-43%. Withdrawal of endosulfan restored the enzyme activity to control level in all the three tissues. The recovery in enzyme activity appears to be due to dissociation of endosulfan or its metabolite(s) from the enzyme molecules and/or fresh synthesis of enzymes. The treatment of actinomycin D or cycloheximide partially inhibited the withdrawal-dependent increase in enzyme activity. This substantiates de novo synthesis of enzyme during recovery period. Since the reduction in enzyme acfvity was more pronounced in response to actinomycin D, endosulfan might be inhibiting the transcription process. But endosulfan did not produce any significant effect on DNA content and RNA/DNA. However, the RNA and protein contents of brain, liver and skeletal muscle decreased significantiy in tissues. The maximum decrease in RNA and protein was approximately 30%-37%. Withdrawal of endosulfan from the medium for 21 days restored the RNA, and protein contents nearly to their control levels. The treatment of actinomycin D or cycloheximide partially inhibited the withdrawal-dependent increase in these macromolecular contents. This effect was more pronounced in case of actinomycin D which again supports the possibility of endosulfan-induced inhibition at transcription level. Conclusion The present study suggests endosulfan-induced impairment of metabolism in fish, which appeared to be due to inhibition of transcription at some unknown points.