In a previous study we provided evidence for the presence in liver of rainbow trout of fatty acid (FA) sensing systems responding to changes in levels of oleate (long-chain FA) or octanoate (medium-chain FA). Since those effects could be attributed to an indirect effect, we have evaluated in the present study in vitro (in the absence of extrahepatic regulatory mechanisms) whether or not liver responds to changes in FA concentration in a way similar to that previously observed in vivo. Accordingly, liver slices were exposed to increased oleate or octanoate concentrations to evaluate changes in parameters related to FA metabolism, FA transport, nuclear receptors and transcription factors, ROS effectors, and glucose metabolism. The responses observed in vitro in liver were in general not coincident with those previously observed in vivo allowing us to suggest that FA sensing capacity of liver in vivo is of indirect nature and could be related among other reasons to an interaction with other endocrine systems and/or to FA sensing in hypothalamus.