We studied fasting and postglucagon plasma C-peptide levels and factors associated with them in two representative studies of middle-aged insulin-treated diabetic patients whose diabetes had been diagnosed after the age of 30 yr. Altogether, 75 men and 79 women from East Finland and 83 men and 62 women from West Finland aged 45-64 yr were studied. Of these patients, 44.4% had undetectable fasting and 38.5% undetectable postglucagon C-peptide concentrations. The phi-coefficient expressing the concordance of fasting and postglucagon C-peptide concentrations in the classification of diabetic patients into nonresponders and responders was .75 in men and .91 in women. In multiple stepwise regression analyses, body mass index (BMI) and the period between diabetes diagnosis and the initiation of insulin treatment were positively and duration of diabetes inversely associated with fasting and postglucagon C-peptide levels in both sexes. We concluded that 1) insulin deficiency is not uncommon in middle-aged insulin-treated diabetic patients whose diabetes has been diagnosed after the age of 30 yr; 2) fasting C-peptide levels contain basically the same information as postglucagon C-peptide levels; and 3) a low BMI, a need for insulin treatment soon after the diagnosis of diabetes, and a long duration of diabetes are predictive of insulin deficiency.