To develop radioimmunoassay for aquaporin-9 (AQP9) and search for its presence in certain rat tissues.Anti-AQP9 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic AQP9 with bovine serum albumin. Radioiodination of AQP9 was performed by chloramin T method followed by purification of radioiodinated material on Sephadex G-25 column.The obtained antibody did not crossreact with other aquaporins, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. AQP9 was extracted from the tissues with acid acetone. The dilution curve of acid acetone extracts of rat liver in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue AQP9 was about 90%, and the intra-assay and inter-assay variations were 4.8% and 7.9%, respectively. AQP9 was found in the liver, testis and brain.These data suggest that this assay system is suitable for the estimation of AQP9 in the tissues.