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In the present study, essential and nonessential metal content and biomarker responses were investigated in the intestine of fsh collected from the areas polluted by mining. Our objective was to determine metal and biomarker levels in tissue respon sible for dietary intake, which is rarely studied in water pollution research. The study was conducted in the Bregalnica River, reference location, and in the Zletovska and Kriva Rivers (the Republic of North Macedonia), which are directly infuenced by the active mines Zletovo and Toranica, respectively. Biological responses were analyzed in Vardar chub (Squalius varda rensis ; Karaman, 1928), using for the frst time intestinal cytosol as a potentially toxic cell fraction, since metal sensitivity is mostly associated with cytosol. Cytosolic metal levels were higher in fsh under the infuence of mining (Tl, Li, Cs, Mo, Sr, Cd, Rb, and Cu in the Zletovska River and Cr, Pb, and Se in the Kriva River compared to the Bregalnica River in both seasons). The same trend was evident for total proteins, biomarkers of general stress, and metallothioneins, biomarkers of metal exposure, indicating cellular disturbances in the intestine, the primary site of dietary metal uptake. The association of cytosolic Cu and Cd at all locations pointed to similar pathways and homeostasis of these metallothionein-binding metals. Comparison with other indicator tissues showed that metal concentrations were higher in the intestine of fsh from mining afected areas than in the liver and gills. In general, these results indicated the importance of dietary metal pathways, and cytosolic metal fraction in assessing pollution impacts in freshwater ecosystems