The mechanisms responsible for the age-related decline in insulin sensitivity have not been clearly identified, but activation of the diacylglycerol/protein kinase C (PKC) signalling pathway (often confined to individual isoforms of PKC) has recently been implicated in the pathogenesis of other insulin-resistant states in both humans and animal models. Fasting serum glucose, insulin and triacylglycerol (triglyceride) concentrations, and results of oral glucose tolerance tests, were compared in groups of 6-week-old (n = 8) and 6-month-old (n = 8) Sprague–Dawley rats. Insulin-responsive tissues (liver, soleus muscle and epididymal fat pad) were collected to compare levels of diacylglycerol, PKC enzyme activity and protein expression of individual PKC isoforms in cytosol and membrane fractions. The older group were heavier (556±14 g, compared with 188±7 g) and relatively insulin-resistant and hyperinsulinaemic (477±73 pM compared with 293±51 pM; P < 0.05) compared with young rats; they also had greater areas under the serum glucose (old, 20.3±1.1; young, 17.3±0.7 mmol·h^-1·l^-1) and insulin (old, 1254±76; young, 721±113 mmol·h^-1·l^-1) profiles following an oral glucose tolerance test, and significantly higher fasting triacylglycerol levels (old, 1.24±0.06 mM; young, 0.92±0.07 mM; P < 0.01). There were no age-related differences in diacylglycerol levels or PKC activity in muscle and liver, but membrane-associated PKC activity was 2.5-fold higher in the adipose tissue of older rats (101±19 compared with 40±5 pmol·min^-1·mg^-1 protein; P < 0.05) due to increased translocation of PKC-β_I, -β_II and -ε. Thus insulin resistance due to normal aging is associated with tissue- and isoform-specific changes in diacylglycerol/PKC signalling. In contrast with diabetes and dietary-induced insulin resistance, there were no changes in diacylglycerol/PKC signalling in skeletal muscle and liver, but isoform-specific translocation and higher PKC activity in adipose tissue may blunt the insulin-mediated inhibition of lipolysis and contribute to the increased triacylglycerol levels observed in older animals.