We studied the effect of chromium (Cr) and lead (Pb) and their combination using the freshwater fish Hoplosternum littorale. After 15 days, we analyzed the responses of multiple biomarkers, such as morphometric and hematological parameters, hepatic and renal enzymes activities (aspartate and alanine aminotransferases, alkaline phosphatase) and oxidative stress markers (antioxidant enzymes activities and lipid peroxidation). Cr caused a decrease in glucose levels and tissue damage in liver and kidney. Pb caused changes in hematological parameters, tissue damage in liver and kidney and induced liver lipid peroxidation. The metal combination modified hematological parameters and increased the hepatic injury biomarkers. According to multivariate statistical analysis, fish exposed to the mixture showed a differential physiologic profile to those exposed to individual metals. Thus, H. littorale has demonstrated to be sensitive for biomarkers response and suitable as potential test species. Our results suggest deleterious effects of sublethal concentrations of heavy metals and support the usefulness of a multi-biomarker approach for the characterization of toxicological mechanisms induced by the exposure to a combination of Cr and Pb.Fil: Ale, Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Bacchetta, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; ArgentinaFil: Cazenave, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentin