In this study, the toxic effects of verapamil (VRP) were studied on juvenile rainbow trout. Oncorhynchus mykiss, by chronic semi-static bioassay. Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 mu g/L) for 0,21 and 42 d. Multiple biomarkers were measured, including morphological indices, hematological parameters and antioxidant responses of different tissues (brain, gill, liver, muscle and intestine). Based on the results, there was no significant change in all parameters measured in fish exposed to VRP at environmental related concentration, but VRP-induced stress in fish exposed to higher concentrations reflected the significant changes of physiological and biochemical responses. Through principal component analysis and integrated biomarker response assessment, effects induced by VRP-stress in each test group were distinguished. Additionally, all parameters measured in this study displayed various dependent patterns to VRP concentrations and exposure time using two-way ANOVA statistic analysis. In short, the multiple responses in fish indicated that VRP induced physiological stress and could be used as potential biomarkers for monitoring residual VRP in aquatic environment; but molecular and genetic mechanisms of these physiological responses in fish are not clear and need to be further studied. (C) 2010 Elsevier B.V. All rights reserved.