Solutions to induce or suppress the initiation of sperm motility in fish have been used to improve reproductive success during artificial fertilization and preservation techniques. The aim of the present study was to evaluate the effects of three solutions (NaCl, glucose, and BTS™) - each prepared with 10 different osmolalities - on the initiation and suppression of fresh sperm motility in Prochilodus lineatus and Brycon orbignyanus. Sperm was diluted in each of the 30 solutions and immediately observed under a light microscope to determine which solutions triggered or suppressed the initiation of sperm motility. When present, motility rate (% motile sperm) was determined at 0, 30, and 120 s post-activation and the motility quality score ranging from 0 (no movement) to 5 (rapidly swimming sperm) was determined at 0 and 30 s post-activation. Osmolality, but not solution composition, significantly affected both motility rate and quality score. Solutions at osmolalities up to 270 mOsm/kg in P. lineatus and up to 180 mOsm/kg in B. orbignyanus induced motility in at least 60% of sperm, with a minimum quality score of 3.0, and were therefore classified as activating agents. The greatest motility at 0, 30, and 120 s post-activation was observed with solutions ranging from 135 to 225 mOsm/kg for P. lineatus and at 135 mOsm/kg for B. orbignyanus. On the other hand, solutions ranging from 360 to 450 mOsm/kg in P. lineatus and 270 to 450 mOsm/kg in B. orbignyanus suppressed motility in at least 95% of sperm and were classified as immobilizing media. The osmolality of the surrounding medium is the key factor in the initiation or suppression of sperm motility in P. lineatus and B. orbignyanus.