Triphenyltin (TPhT) is used worldwide in pesticide formulas for agriculture. Toxic effects of this compound to aquatic life have been reported; however, the biochemical response of fish exposed to different concentrations of TPhT hydroxide (TPhTH) was investigated for the first time in this study. The lethal concentration (LC50) of TPhTH to silver catfish, Rhamdia quelen, was calculated from an acute-exposure experiment (96 h). In addition, acethylcholinesterase (AChE) activity in brain and muscle-as well as glucose, glycogen, lactate, total protein, ammonia, and free amino acids in liver and muscle-were evaluated in a chronic-exposure experiment (15-day exposure). Speciation analysis of tin (Sn) was performed in fish tissues at the end of both experiments using gas chromatography coupled to a pulsed-flame photometric detector (GC-PFPD). Concentrations of TPhT, diphenyltin, and monophenyltin (reported as Sn) were lower than limits of quantification (10σ criteria). Waterborne TPhTH concentration used through the experiment was also evaluated by GC-PFPD, and no degradation of this species was observed. The LC50 value for silver catfish juveniles was 9.73 μg L(-1) (as Sn). Decreased brain and muscle AChE activities were observed in fish exposed to TPhTH in relation to unexposed fish (control). Liver glycogen and lactate levels were significantly higher in fish kept at the highest waterborne TPhTH concentration compared with the control. Liver and muscle glucose levels of fish exposed to all TPhTH concentrations were significantly lower than those of control fish. Silver catfish exposed to all TPhTH concentrations showed lower total protein values and higher total free amino acids levels in liver and muscle compared with controls. Total ammonia levels in liver and muscle were significantly higher for the highest TPhTH concentration compared with controls. In conclusion, TPhTH caused metabolic alterations in silver catfish juveniles, and the analyzed parameters can also be used as bioindicators for TPhTH contamination.