To avoid repeated production of 125I tissue mash standards and to decrease inter-experiment variability, a set of commercially available, high activity plastic 3H standards was calibrated for use with 125I autoradiographs. Standards with a range of 2.0-7390 microCi 3H/g plastic were used since they produced autoradiographs of suitable optical density (darkness) at exposure times of 4 to more than 72 h. Tissue mash standards at 12 concentrations of 125I were produced by homogenizing rat brains and adding 125I. These were frozen and sectioned at thicknesses ranging from 6 to 120 microns and mounted on microscope slides. Each set of tissue standards, along with slides bearing the plastic 3H standards, was opposed to film for various times. The calibration of the 3H standards was done separately for each time point and section thickness. Autoradiographs were analyzed using a computer assisted image analysis system. The standard curve generated for each set of 125I-labelled tissue standards was correlated with the 3H standards. It was found that the relationship between the 3H plastic standards and the 125I tissue mash standards was not simple and depended on exposure time, thickness of the section, as well as the concentration of radioactivity in a given standard. However, this accurate calibration of the 3H standards yields long-lived standards for 125I autoradiography useful over a wide range of radionuclide concentrations, tissue thicknesses and exposure times.