A radioimmunoassay method suitable for measuring levels of B-endorphin, B-lipotropin and proopiocortin in tissue and plasma extracts was developed and the method was evaluated by using 3 independently prepared antisera. Of the several variables tested the choice of assay buffer and test tubes and the purification of tracer were found to be the most critical in the successful performance of B-endorphin radioimmunoassay. The prevention of degradation of tracer during incubation was also necessary when crude tissue extracts or plasma were assay. The sensitivities of the assays obtained with the 3 antisera utilized (Bendo 2, K2 and RB 100) were 1, 2.8 and 4 fmol B-endorphin per tube. All the antisera crossreacted equimolarly with B-lipotropin. The method was employed to measure the levels of B-endorphin immunoreactivity in rat pituitary and plasma by separating the different immunoreactants by gel filtration. It was found that both pituitary and plasma contain significant amounts of proopiocortin, B-lipotropin and B-endorphin, the molar proportions being 10:33:57 in pituitary and 15:15:17 in plasma, respectively. Both anterior and posterior lobes of rat pituitary were found to contain all the three immunoreactants. However, anterior lobe contained mostly the larger molecules, while posterior lobe was rich in B-endorphin. No absolute levels of the immunoreactants could be obtained due to the heterologous system used. Moreover the elution pattern of the immunoreactivity was found to be dependent on the conditions used for elution in gel filtration: higher proportion of the immunoreactivity eluted like B-endorphin when the elution was done in dissociating conditions (6 M urea) compared to elution with ordinary buffers.