Official documents and archives suggest that the alien crayfish Pacifastacus leniusculus (Dana, 1852) was introduced into Japan from North America on five occasions from 1926 to 1930, and populations have survived in Hokkaido, Nagano, and Shiga Prefectures. The chemical conditions and biological environment of an established habitat near their original a release point in Akashina, Azumino City, Nagano Prefecture, were surveyed on four occasions in 1998 and 1999. Water quality (pH, DO, BOD) and WT were recorded seasonally at each of eight stations, and crayfish and other aquatic macro-organisms were collected. Some predatory fishes (Oncorhynchus mykiss, Anguilla japonica, and Lepomis macrochirus) that might actively eat juveniles of P. leniusculus were recorded. Individual density of P. leniusculus was relatively low compared to its other habitats in Japan. Only 52 crayfish were collected during this study, on 19 of 32 sampling occasions (eight sites, four times each). The shape of the acumen and the species composition of ectosymbiotic crayfish worms (Annelida: Clitellata: Branchiobdellidae) can be used as tools to trace the origin of introduced of P. leniusculus in Japan. The present crayfish from Nagano Prefecture have a relatively short acumen, different from those of specimens from Shiga and Hokkaido Prefectures. Furthermore, the crayfish worm Xironogiton victoriensis occurs only on crayfish from Akashina, Nagano Prefecture. The dates of introduction of P. leniusculus in Nagano (1926, 1929) also differ from those for Hokkaido (1930) and Shiga Prefecture (1926). These findings suggest that the regional population of P. leniusculus in Nagano has a different origin than those in Shiga and Hokkaido, having originated from crayfish that were introduced independently from North America.