The particular characteristics of fish embryos require the development of specific methods for cryopreservation. One of the main obstacles is related to the presence of membranes and compartments with different water and cryoprotectant permeability. To assess dimethyl sulfoxide (Me2SO4) permeability, we exposed turbot embryos (Scophthalmus maximus) at F stage (tail bud) to the cryoprotectant solutions used in a vitrification protocol and then evaluated the Me2SO4 content inside the embryo using high-performance liquid chromatography (HPLC). The Me2SO4 influx was analyzed in normal embryos and in embryos treated with pronase (2mg/ml) in order to increase chorion permeability. The evaluation was made after each step of cryoprotectant incorporation and removal. Three embryo compartments were distinguished: the perivitelline space (PVS), the yolk sac (YS) and the cellular compartment (CC), and the relative volumes of each, estimated using stereoscopic microscopy imaging, were 11.37, 81.23 and 7.40%, respectively. The Me2SO4 concentration inside the embryos was calculated based on their entrance into one, two or three compartments. Results suggest high entrance of Me2SO4 into the PVS and a low concentration of this cryoprotectant inside the other compartments. Pronase did not significantly increase Me2SO4 influx, but facilitated its elimination during the washing steps.