The aspidogastrid Cotylaspis insignis is normally found as an ectoparasite at the junction of the gills and visceral mass in freshwater mussels from a variety of species. It is not known if the osmotic environment at this site more closely approximates that found in stream/pond water or mollusk blood/hemolymph. The objectives of this study were to assess the in vitro effect of osmolality on adult C. insignis egg production and survivorship over 3 and 14 d, respectively, at 20°C. Worms recovered from the fat mucket, Lampsilas radiata luteola, and maintained in artificial snail water/hemolymph (ASW; 102 mOsm) showed a trend for greater survivorship than in artificial pond water (APW; 15 mOsm), though a significant difference was only noted on day 2 postexposure (PE). The mean ± SE eggs shed from adult worms in the controlled osmotic conditions over days 1–3 PE exceeded the number of eggs initially present within adults on day 0, indicating that egg production continued by adult worms in vitro. Egg production was much reduced after day 1 PE, but worms released significantly more eggs in ASW than in APW on days 2 and 3 PE. These results suggest that the osmolality at the gill visceral mass junction more closely approximates that of ASW rather than APW.