The results of studies on the activity of lysosomal enzymes in different organs (liver, gills, muscles, gonads) of mature threespine stickleback Gasterosteus aculeatus L. at the beginning and in the end of the spawning period are reported. Fish were captured from three spawning grounds in the Gulf of Kandalaksha, White Sea, which differed in temperature, salinity, retention time, aquatic vegetation and other parameters. The samples were analyzed for the activity of five acid hydrolases (acid phosphatase, RNase, DNase, β-galactosidase, β-glucuronidase). During the spawning period, fish were shown to be in a state of high metabolic stress, as evidenced by an increase in acid phosphatase and glycosidase activity in the liver and especially a significant increase in the level of almost all the studied enzymes in the skeletal muscles of stickleback males and females from all habitats by the end of spawning. We are thus driven to the conclusion about a powerful effect of endogenous factors (sex steroid and other hormones) on fish metabolism. As they hydrolyze intracellular reserves and the components which are less necessary at the moment, lysosomal enzymes provide the body with important energetic and constructive materials. Lysosomal enzymes take part in the transport of substances from internal organs to gonads to promote their maturation in the fish arriving at spawning grounds later, and help compensate the lack of exogenous nutrients, especially in stickleback males who care for the offspring in the end of the spawning period. The elevated activity of acid hydrolases in gonads of females is apparently associated with the resorption of unspawned eggs and regeneration of gonad tissues. Gills, unlike other organs, featured an inhibition of the activity of glycosidases and activation of RNase by the end of the spawning period even in the spawning ground with the most favorable conditions (Seldyanaya Bay), which is most likely due to a sharp change in salinity in this area over the spawning period. In general, the response of lysosomal hydrolases to environmental fluctuations in different habitats was less pronounced compared with the effect of endogenous factors associated with spawning.