Organic acids such as citric acid enhance the vaginal absorption of luteinizing hormone-releasing hormone, the potent analogue (leuprolide), and insulin. The mechanism of this absorption enhancement was investigated with leuprolide and hydrophilic markers such as phenol red and Evan's blue. The absorption of the analogue was increased by lowering the pH of the solution and increased more by adding citric, succinic, tartaric, and malonic acids. The absolute bioavailability after vaginal administration of the 5% citric acid solution was 16.7% at pH 3.5 and 38.4% at pH 1.8. The enhancing potency of the absorption correlated well with the chelating ability of the organic acids. The vaginal absorption of phenol red was also enhanced with citric acid and edetic acid, but the enhancing effect of edetic acid was eliminated by adding equimolar calcium ion. These results suggest that the acidifying and chelating abilities of the acids may result in a potent enhancement of the vaginal absorption of leuprolide. A leakage experiment using Evan's blue on the vaginal membrane indicated that the blood-vaginal epithelium barrier was loosened with the administration of citric acid; this change was overcome rapidly.