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Microhabitat and niche segregation of coexisting two freshwater snail species, Semisulcospira libertina (Gould, 1859) (Prosobranchia ; Pleurocerdiae) and Clithon retropictus (Matens, 1879) (Prosobranchia ; Neritidae) were studied. Semisulcospira libertina, which is an ovoviviparous snail, is distributed in freshwater in Japan. Clithon retropictus, which is an oviparous snail, is distributed in brackish water and freshwater of south-east Japan. These two species show sympatric distribution at fresh water area in the middle reaches of the Goino River (31°28′N, 130°31′E), Kagoshima City, Kyushu Island, Japan. In this study, we showed that S. libertina and C. retropictus segregated their niche in the sympatric area. We settled four places of investigation areas by the river mouth from the upper reaches of the Goino river and each named as station 1, 2, 3 and 4. Samples were collected at random every month in these four stations and population densities were estimated. Collected snails were measured by shell width. After measurement, snails were released again. In the S. libertina population, the frequency distributions of shell size showed that newborn juveniles mainly appeared in April and December. In the C. retropictus population, the frequency distributions of shell size showed that newborn juveniles appeared in September. Salinity-tolerance experiment of C. retropictus showed that adults were tolerant of the freshwater condition compared with juveniles. In the investigation of microhabitat, the type of microhabitat was divided by riffle-pool type and by substratum type, and four types (pool-stone, pool-sand, rapid-stone and rapid-sand) of microhabitat were defined. At the time of survey, twenty quadrats (50 cm×50 cm) were set in each of type of microhabitat in the four stations randomly, and the number of snails appeared in the quadrates were counted to estimate the microhabitat preference of two species. This measurement was investigated in August (the summer season), October (the fall) and December (the winter) to examine whether there was a seasonal change of microhabitat preference. Semisulcospira libertina preferred pool to rapid. Adults of Clithon retropictus preferred rapid to pool and preferred stone-flat to sand-flat. In the Station 1 where only S. libertina was distributed alone, most S. libertina inhabited stone-flat surface. On the other hand, in the Station 2 and Station 3 where S. libertina coexisted with C. retropictus, S. libertina rarely inhabited stone-flat surface. In the Station 4 where only C. retropictus inhabited alone, C. retropictus had a tendency to prefer stone-flat surface as well as in the Station 2 and the Station 3 where C. retropictus coexisted with S. libertina. From these facts, it is concluded that S. libertina narrows its niche by limiting using microhabitat type where they coexist with C. retropictus. In other words, at the place where S. libertina coexists with C. retropictus, some competitions occur between the two species, and the niche is segregated by S. libertina evacuating from stone-flat as the life space.