Using gelatin‐SDS‐PAGE, proteolytic activity was found in the seminal plasma of 10 teleosts: common carp Cyprinus carpio, bream Abramis brama, ide Leuciscus idus, chub Leuciscus cephalus, rainbow trout Oncorhynchus mykiss, grayling Thymallus thymallus, perch Perca fluviatilis, pike Esox lucius, goldfish Carassius carassius and pikeperch Stizostendion lucioperca. This activity was also measured, using azoalbumin as a substrate, in the seminal plasma of these species, with exception of pikeperch and goldfish. When azoalbumin‐hydrolysing activity was expressed per volume, it was highest in common carp. Otherwise, as expressed per g of protein, the activity was highest in pike. The lowest proteolytic activity (expressed per g and volume) was observed in perch seminal plasma. Using gelatin containing polyacrylamide gels for detecting gelatinolytic activity, species‐specific electrophoretic profiles were found. For all cyprinids two similar bands with a molecular mass of 68 and 74 kDa were found. The seminal plasma of grayling and rainbow trout showed similarities in the 41 kDa band. Perch and pikeperch had one similar main band with a molecular mass of 61 kDa. Proteolytic enzymes of seminal plasma from pike showed high individual variability. These results suggest that multiple forms of proteolytic enzymes exist in seminal plasma of teleosts and they differ among fish families and species.