AbstractRhodamine‐123 (Rh123) was iodinated with123I in presence of Iodogen. Yield of the labelled Rh123 varied greatly depending upon various reaction parameters. Optimum reaction conditions with respect to molarity and pH of the buffer, reaction time, amounts of iodogen, carrier iodide and Rh123, were determined through carefully controlled reactions. Radiochemical yields of 50–60% were obtained within less than 2 hrs. using 0.05M NH4H2PO4buffer at pH5, 200 μg iodogen, 250 μg Rh123 and no‐carrier‐added iodide. Separation of the labelled Rh123 in greater than 98% radiochemical purity was achieved by flash chromatography on silica gel. The overall isolated yield of radioiodinated Rh123 ranged from 25–35% with respect to the added radioiodide. TLC and HPLC methods were developed for analysis of the labell