Studies regarding the effects of extender composition, osmolality, cryoprotectant (CPA) and equilibration time on the induction/suppression of sperm motility are necessary to establish standard activating agents and immobilizing media for improving both artificial fertilization and preservation techniques. Thus, the aim of this study was to evaluate the effects of these factors on fresh sperm motility in piracanjuba (Brycon orbignyanus) and streaked prochilod (Prochilodus lineatus). Twenty four media, as a combination of six extenders (BTS™ and glucose solutions at 270, 315 and 360 mOsm/kg) with the CPAs DMSO, methanol, methyl glycol (MG) and a control without CPA, were prepared. Immediately after dilution, samples were observed under a light microscope to confirm whether different extender-CPA combinations would suppress the initiation of sperm motility. Motility was then triggered in 92 mOsm/kg NaCl and evaluated immediately after dilution (non-equilibrated samples) and after a 30-min equilibration time at 4°C for motility rate and motility quality score (0 = no movement; 5 = rapidly swimming sperm). In both species, motility was initiated in all samples diluted in BTS-270-control, Glu-270-MG, Glu270-control and in all combinations containing DMSO. In B. orbignyanus, motility rate (77 to 92%) and motility quality score (3.3 to 4.7) of non-equilitrated samples was not significantly affected by any parameter. After 30 min, however, motility quality score decreased in most of the samples, mainly when diluted in BTS™ (3.3 to 4.2). In P. lineatus, motility rate was significantly higher in non-equilibrated samples (overall mean = 83%) compared to 30-min equilibrated samples (overall mean = 75%). Motility quality score of nonequilibrated samples was not affected by any parameter (3.3 to 4.2), but samples equilibrated in DMSO yielded the lowest score (3.0). Sperm motility (rate and score) was affected differently in B. orbignyanus compared to P. lineatus, and this finding should be considered when developing a methodology for sperm cryopreservation.