The present work aimed to study in rats whether substitution of a low level of fish oil (FO; 2.2% of calories) into a low-fat diet (6.6% of calories from fat as peanut-rape oil or control diet) 1) has a tissue-specific effect on insulin signaling pathway and 2) prevents dexamethasone-induced alteration of insulin signaling in liver, muscle, and adipose tissue. Sixteen rats were used for study of insulin signaling, and sixteen rats received an oral glucose load (3 g/kg). Eight rats/group consumed control diet or diet containing FO over 5 wk. Four rats from each group received a daily intraperitoneal injection of saline or dexamethasone (1 mg.kg(-1).day(-1)) for the last 5 days of feeding. In liver, FO decreased phosphatidylinositol 3'-kinase (PI 3'-kinase) activity by 54% compared with control diet. A similar result was obtained in muscle. In both liver and muscle, FO clearly amplified the effect of dexamethasone. FO did not alter early steps of insulin signaling, and in muscle GLUT4 protein content remained unaltered. In adipose tissue, FO increased PI 3'-kinase activity by 74%, whereas dexamethasone decreased it by 65%; inhibition of PI 3'-kinase activity by dexamethasone was similar in rats fed FO or control diet, and GLUT4 protein content was increased by 61% by FO. Glycemic and insulinemic responses to oral glucose were not modified by FO. In conclusion, FO increased PI 3'-kinase activity in adipose tissue while inhibiting it in liver and muscle. The maintenance of whole body glucose homeostasis suggests an important role of adipose tissue for control of glucose homeostasis.