We evaluated electrolytic separation factors of hydrogen isotopes by SPE (Solid Polymer Electrolyte) for application to environmental tritium analysis. The apparent separation factors a for deuterium and a for tritium were determined as 3.5 ± 0.1 and 6.2 ± 0.5, respectively. The tritium enrichment of 8.4 times was achieved, when a 1000 ml of sample water was electrolyzed to about 60 ml. The chemical composition changes before and after the electrolysis were examined, showing an increase in H+ and Na+ concentrations and a decrease in Mg2+ and Ca2+concentrations. F-, which was not contained in the sample water, was detected after electrolysis accompanying with a reduction of SO42-, Cl- and NO3-. The memory of tritium and ions in the electrolysis cell after electrolysis was possible to be eliminated by washings with de-ionized water. Tritium concentrations of rain at Kumamoto, Japan were determined with a combination of the present electrolytic enrichment system and liquid scintillation counting.