Sperm of most fresh water teleosts become motile when released into the hypotonic fresh water environment, but the role of osmolality and Ca2+ on sperm motility is not clear. Osmotic pressure and Ca2+ concentrations increase from fresh water to brackish water. Java carp Puntius javanicus and catfish Clarias batrachus live and reproduce only in fresh water. On the other hand, goby Oxyeleotris marmorata can acclimate and reproduce from fresh water to brackish water. In the present study, sperm motility and trajectory were compared among these three Indonesian endemic species. Sperm of Java carp, goby, and catfish begun to move in the hypotonic condition (< 200 mOsm/kg). However, the response to Ca2+ was different among these teleosts. In the presence of Ca2+, Java carp sperm swam in circular paths and immediately become quiescent, suggesting that Java carp sperm motility is activated in hypotonic aquatic environment without Ca2+. Goby sperm swam straightforward in the presence or absence of Ca2+. Percentages of motile sperm increased in 100-200 mOsm/kg but suppressed by removal of Ca2+. Regarding sperm motility and trajectory, no response was found in catfish sperm. These results suggest that a response to Ca2+ is different among sperm of the three species and suited to their habitat.