Abstract The interactions of 54Mn radionuclide with humic (HAB) and fulvic acid (FAB) isolated from freshwater sediment of the Butoniga impoundment were studied in a freshwater system from Butoniga impoundment, and in a diluted seawater system from the Rašla Bay at elevated pH by high-voltage paper electrophoresis. In this way, it was possible to detect cationic, anionic and immobile 54Mn species, and to calculate their electrophoretic mobilities. The interaction of 54 Mn with humic substances (HS) in a freshwater system is demonstrated by the formation of an electrophoretically immobile zone and the formation of the cationic forms of 54Mn having smaller electrophoretic mobilities than cationic forms of 54Mn in a freshwater system without HS added. The amount of cationic forms decreases in the presence of HS. This effect is enhanced with an increase in HS concentration in the range 5–100 mg l −1 and in an increase in pH from 9.5 to 10.5. The addition of HAB up to 100 mg l −1 and the increase of pH from 7.9 to 9.0 in diluted seawater systems do not cause significant changes of cationic electrophoretic mobilities of 54Mn, nor the formation of an electrophoretically immobile form of 54Mn.