The milt of individual males of Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus) was frozen without cryoprotector (at -79°C) or cryopreserved with methanol as the cryoprotector. The activity of arylsulfatase (AS), acid phosphatase (AcP), β-N-acetylglucosaminidase (NAGase), and protein concentration was determined. The protein concentration and enzymatic activities in supernatant obtained after cryopreservation were higher than in milt plasma, but they were lower than that in the material obtained after freezing at -79°C. The protein and enzymatic leakage of sterlet spermatozoa was statistically higher in supernatants that had been frozen at-79°C than in those that had undergone cryopreservation. Differences in the protein and AS leakage the Siberian sturgeon supernatants were also observed.