In the present paper we determined low molecular heavy metal binding protein called metallothionein (MT) in fish spermatozoa sampled from four fish species (Acipenser baerii, Acipenser ruthenus, Perca fluviatilis and Sander lucioperca). The spermatozoa samples were analyzed with differential pulse voltammetry DPV (Brdicka reaction). The average content of MT in the fish sperm was 0.64 ± 0.55 μm. The highest content of MT was determined in spermatozoa of S. lucioperca (0.96 ± 0.86 μm), followed by P. fluviatilis (0.88 ± 0.11 μm), A. ruthenus (0.57 ± 0.60 μm) and A. baerii (0.43 ± 0.25 μm). The differences in the content of MT was probably related to the differences in age and perhaps to the health status based on the environmental conditions in which the tested specimens developed while also other still unknown factors may have been involved.