Lipoprotein lipase (LPL) is considered as a key enzyme in the lipid deposition and metabolism of many tissues. Information on LPL activity and its regulation in fish remains very scarce. In the present study, we have examined the nutritional regulation of LPL activity by conducting post-feeding and fasting experiments in rainbow trout (Oncorhynchus mykiss). As insulin plays an important role in the nutritional regulation of LPL activity in mammals, the effects of this hormone were tested in vivo by intraperitoneal administration. Moreover, we conducted in vitro studies using fat pads of rainbow trout to better clarify the direct role of insulin and tumor necrosis factor-alpha (TNFalpha) as possible regulators of LPL activity in rainbow trout. LPL activity in adipose tissue increased in response to feeding, 4h after ingestion of food, then decreasing to basal levels at 6h. No clear response was found in either red or white muscles, where LPL values were lower. Moreover, fasting produced a down-regulation of LPL activity in adipose tissue, concomitant with low levels of plasma insulin. While insulin administration stimulated LPL activity of adipose tissue 3h after injection, no response was observed in red or white muscles. Finally, in vitro studies using fat pads revealed that insulin significantly stimulated the proportion of LPL in active conformation at the extracellular level. On the other hand, TNFalpha did not greatly affect LPL activity using this in vitro model. These data indicate that LPL activity is regulated in a tissue-specific manner following food intake, and suggest that insulin is an important regulator of LPL activity in the adipose tissue of rainbow trout.