BACKGROUND: Cryopreserved semen could facilitate procedures during the artificial reproduction in fish. Factors affecting cryopreservation efficiency are important to define efficient protocols.This study investigated the application of cryoprotectants on the quality of piracanjuba fish semen, the sperm concentration required for oocyte fertilization and spermatic activation.We evaluated two intracellular cryoprotectant solutions (DMSO and methanol) and two extracellular cryoprotectant solutions (egg yolk and lactose) to cryopreserved piracanjuba semen. Sperm motility rate, motility duration and spermatic alterations were assessed.The protocol for piracanjuba semen cryopreservation can use solutions including either DMSO or methanol as intracellular cryoprotectant and egg yolk or lactose as extracellular cryoprotectants.