The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue-and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n= 24) and lean (fa/-, n= 24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (4887 vs 3159g) with fasting hyperglycaemia (10· 50· 8 vs 7· 70· 1 mM) and hyperinsulinaemia (7167363 vs 25162 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (17416 vs 10812 pmol/min/mg protein, P< 0· 05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-θ and PKC-ε: eg PKC activity in the membrane fraction of muscle from obese animals was 28342 (fed) vs 10720 pmol/min/mg protein (fasting) compared with 19727 (fed) and 15421 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-θ and PKC-ε. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes.