Diabetes mellitus is often associated with lipid abnormalities that may differ with sex. In this work we studied biliary lipid secretion in male and female anaesthetized Wistar rats (250 g). Diabetes was induced by a single intraperitoneal injection of streptozotocin (6 mg/100 body weight) 6 days before carrying out the studies on bile secretion. Our results confirm the existence of sex differences in bile formation and composition, most of them probably due to a higher (+ 27%) bile acid output in the female animals. Diabetes induced profound alterations in these sex differences. (a) Bile flow was reduced in both sexes, but more markedly so in female diabetic rats; thus the difference observed in healthy animals was reduced (from 2.22 to 1.58 and from 1.84 to 1.40, μl/min per g liver in female and male rats, respectively). (b) Bile acid and phosphatidylcholine outputs were increased to a similar extent (bile acid output: from 46.7 to 55.8 nmol/min per g liver, in females and from 36.8 to 50.7 nmol/min per g liver, in males; phosphatidylcholine output: from 3.3 to 13.1 nmol/min per g liver, in females and from 4.5 to 12.5 nmol/min per g liver, in males), and hence the sex differences were abolished. (c) Cholesterol output was increased in both sexes, but this enhancement was significantly higher in female rats (from 0.75 to 1.31 and from 0.65 to 0.89 nmol/min per g liver, in females and males, respectively). (d) The fractional pool of phospholipid species secreted into bile was different in female compared with male rats. The percentage of phosphatidylcholine was higher in female than in male healthy rats. Streptozotocin treatment reversed this proportion, which suggests that changes in the phospholipid composition of the canalicular plasma membrane may play a role in the observed alterations in biliary lipid secretion during diabetes mellitus. Most of the above-described streptozotocin-induced changes were prevented by insulin replacement from the 3rd to the 6th days after streptozotocin injection. In summary, the present study describes alterations in sex differences in biliary lipid secretion of strepetozotocin-induced diabetes. These changes are dependent on the insulin deficiency state rather than on a direct hepatotoxicity of the diabetogenic drug.