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The absorption of insulin across the vaginal mucosa into the systemic circulation was studied in ovariectomized rats given subsequent estrogen treatment. Blood glucose levels were determined as an indirect measure of insulin absorption, and the effect of various enhancers on the hypoglycemic response was investigated. In the absence of any enhancer, no decrease in blood glucose levels was observed after vaginal administration of insulin. However, the coadministration of sodium taurodihydrofusidate, polyoxyethylene-9-lauryl ether, lysophosphatidylcholine, palmitoylcarnitine chloride, and lysophosphatidylglycerol significantly increased hypoglycemia, whereas citric acid had little effect. The histological changes in the vaginal epithelium after treatment with the enhancer systems were variable and often severe. While the efficacy of these compounds in promoting the vaginal absorption of insulin is encouraging, their mechanisms of action and long-term histological effects are yet to be defined.