Radioactive iodine (131I and 123I) labelled methylene blue is used for the early diagnosis of melanoma metastases. We studied the iodination reaction of methylene blue (using "cold" iodine) in order to characterise the iodination product(s) as far as number and position of iodine atoms introduced on the aromatic ring(s) is concerned. The reaction was carried out under the same experimental conditions used for the radioactive one, that is in a large excess of methylene blue. The ionspray HPLC-MS analysis of the reaction mixture showed that the iodinated methylene blue was present only in a very small amount and the main iodinated product was a demethylated one, coming out from the iodination of an impurity azure B. We also studied the iodination reaction of azure B in order to better explain the reaction pathway. Commercial azure B contains impurities of methylene blue and all the possible demethylated derivatives. HPLC-MS analysis of the reaction mixture allowed a complete characterisation of the iodinated and bis-iodinated products.