The need for defining the assay conditions assumes greater significance for the manufacturer of RIA kits. This stems from the consideration that despite high quality of kit reagents in the kits, it is the definition of optimum conditions of assay that makes them effective and also permits to evaluate the limits of the sensitivity and precision. In this connection we have studied in detail a number of parameters mainly to define the stability of the assay system to changes in the protein concentration, the influence of polyethylene glycol in various ionic media, incubation parameters for systematic error analysis in three separation systems based on dextran coated charcoal, plain polyethylene glycol and polyethylene glycol assisted second antibody. Second antibody in a preprecipitated form was found to be readily adaptable as a reagent. Among the three methods of separation studied, the second antibody employed as a complex with primary antibody assisted by polyethylene glycol had superior separation without any specific criticality, characteristics as judged by the variations at various dose levels over an extended period and provided a practical procedure for routine assays.