Perfluoroalkyl substances (PFHxS, FHUEA, PFOA, PFOS, FOSA, N-methyl FOSA and PFNA) from seven sites on the Svitava and Svratka rivers in the Brno conurbation (Czech Republic) were determined in fish blood plasma and water. Concentrations of PFHxS, FHUEA, FOSA, and N-methyl FOSA were below detection limits. Major compound in fish blood was PFOS (38.9-57.8 ng mL(-1)), followed by PFNA and PFOA. In water, the major compound detected was PFOA (1.7-178.0 ng mL(-1)), followed by PFOS and PFNA. A significant (p < 0.05) correlation for PFOA concentration in blood plasma and water was found (r = 0.74).