A14‐Iodoinsulin is a close biochemical and pharmacological mimic of insulin. Human insulin was labelled in its A chain tyrosine‐14 residue by direct iodination with the positron‐emitter iodine‐124 (β+=25.6%; t1/2=4.15 days) to provide a radiotracer for imaging with positron emission tomography (PET). Several reagents were compared for conversion of cyclotron‐produced [124I]iodide into a reactive species for the labelling reaction. Radiochemical yields from the use of Iodo‐Gen®, Chloramine‐T, N‐bromosuccinimide or lactoperoxidase–hydrogen peroxide were similar [35% (n=1), 33±9% (n=10), 32±11% (n=25) or 33% (n=2), respectively]. [124I]A14‐Iodoinsulin was separated from unreacted insulin and radioactive by‐products by tandem reverse phase HPLC and rapidly formulated for intravenous injection by adsorption on a Sep‐Pak tC18‐Plus® cartridge, followed by elution with 10 mM hydrochloric acid–ethanol (1:1 v/v, 1 ml). This radiotracer can now be obtained in useful radioactivities at high effective specific radioactivity and is now being applied to PET studies of its biodistribution in living subjects. Copyright © 2001 John Wiley & Sons, Ltd.