Zebra mussels have dispersed from their original site of introduction in the Great Lakes into the Mississippi River, Hudson River, and other watersheds in which they will encroach upon brackish water estuaries. To investigate their likelihood of reproductive success in such estuaries, we investigated the conditions of temperature, salinity, and acclimation under which spawning and fertilization could occur. Reproductive function of mussels that were acclimated to salinities up to 7.0 parts per thousand (ppt) at 12°, 20°, and 27°C for 1 to 21 days was tested. Reproductive function of non-acclimated mussels that had been maintained in fresh-water aquaria was also tested in various salinities. Spawning was induced by serotonin, previously demonstrated to elicit spawning of fertile gametes in fresh water. Successful fertilization was indicated by oocyte cleavage after adding sperm. Nonacclimated mussels spawned in salinities of 1.75 and 3.5 ppt at 12°, 20°, and 27°C, but not at 7.0 ppt. Fertilization using gametes from non-acclimated mussels occurred only in fresh water and at 1.75 ppt. Acclimation for as little as 2 days enhanced spawning. Fertilization rate in a salinity of 3.5 ppt improved within 4 days of acclimation and continued at a high level for as long as 21 days of acclimation. Although animals acclimated for 4 days in 3.5 ppt spawned readily when tested in salinities as high as 7.0 ppt, almost no fertilization occurred in 7.0 ppt. The reduction in fertilization at increasing salinities may be due in part to reduced sperm motility. Unfertilized oocytes remain intact for hours in fresh water; however, in salinities as low as 0.7 ppt, unfertilized oocytes tended to rupture within 2 hours. These data show that although sudden increases in salinity produce an immediate decrease in the reproductive capacity of zebra mussels, acclimation to brackish water can occur, and zebra mussels may be able to reproduce in brackish water below 7.0 ppt.