The 28-kd plasma protein α1-microglobulin is found in the blood of mammals and fish in a free, monomeric form and as high-molecular-weight complexes with molecular masses above 200 kd. In this study, iodine 125–labeled free and high-molecular weight rat α1-microglobulin (a mixture of α1-microglobulin/α1-inhibitor-3 and α1-microglobulin/fibronectin complexes) were injected intravenously into rats. The distribution of the proteins was measured by using scintillation camera imaging. Both forms of 125I-labeled α1-microglobulin were rapidly cleared from the blood, with a half-life of 2 and 16 minutes for the initial and late phase, respectively, for free α1-microglobulin; and a half-life of 3 and 130 minutes for the initial and late phase, respectively, for the complexes. After 45 minutes, 6%, 16%, 27%, 13%, and 34% of the free 125I-labeled α1-microglobulin and 18%, 21%, 6%, 10%, and 42% of the 125I-labeled α1-microglobulin complexes were found in the blood, gastrointestinal tract, kidneys, liver, and the remainder of the body, respectively. The local distribution of injected 125I-labeled α1-microglobulin in intestines and kidneys was investigated by microscopy and autoradiography. In the intestine, both forms were distributed in the basal layers, villi, and luminal contents. The results also suggested intracellular labeling of epithelial cells. Well-defined local regions containing higher concentrations of injected protein could be seen in the intestine. In the kidneys, both forms were found mostly in the cortex. Free 125I-labeled α1-microglobulin was found predominantly in epithelial cells of a subset of the tubules, whereas the 125I-labeled complexes were more evenly distributed. Intracellular labeling was indicated for both α1-microglobulin forms. The results thus indicate a rapid transport of 125I-labeled α1-microglobulin from the blood to most tissues. (J Lab Clin Med 2001;137:165-75).