To optimize the techniques of captive breeding of flounder Paralichthys adspersus, a methodology was developed for the cryopreservation of spermatozoa of this species. The effect on sperm motility post-thawing, using three different concentrations (1.0; 1.5 and 2.0 M) of DMSO as cryoprotective agent and five different freezing rates -7.5; -10; -12.5; -20 and -30 degrees C min(-1), with an automatic programmable freezer was evaluated. The highest percentages of post-thawing sperm motility were obtained by freezing sperm at -10 degrees C min(-1), no significant differences (P < 0.05) were founded between the three different concentrations of DMSO used. Subsequently, we evaluated the effect of a non-permeable cryo-additive (chicken egg yolk, VHG), in order to obtain an increasing of the percentage of sperm motility. We used a cryoprotectant solution including DMSO at three different concentrations adding 10% VHG v/v. The highest percentages of sperm motility (71.71 +/- 13%) were obtained at the freezing rate of -10 degrees C min(-1) without significant differences between the three concentrations of the cryoprotectant solution in which the sample was incubated sperm (P < 0.05). A high significant difference between the sperm motility percentages post-thawing using DMSO and DMSO with VHG, was observed.