This paper reports a radioimmunoassay method for triiodothyronine (T3) and the application of this assay to the study of plasma and tissue T3 concentration in the rat. Several antisera formed to a T3-bovine fibrogen complex in guinea pigs and T3-thyroglobulin complex in rabbits are shown to have low or no cross reactivity with T4, monoiodotyrosine, diiodotyrosine, tetraiodothyroacetic acid (TETRAC) and reverse T3. Cross reactivity with T3 derivatives, triiodothyroacetic acid (TRIAC), and triiodothyropropionic (TRIPROP) was variable, some antisera differentiating moderately well and others not at all. An extraction method is described which removes approximately 85% of added 125I-T3 or unlabelled T3 from tissues and in the final step represents 57% of total tissue T3. Mean plasma T3 in 5 normal male rats was 58 plus or minus 6.0 ng/100 ml (SEM), in liver 769 plus or minus 56 ng/100 g, and kidney 627 plus or minus 39 ng/100 g. Tissue to plasma concentration gradients for liver and kidney were 13.3 and 10.8, respectively.