Recombinant murine gamma-interferon (rIFN-gamma) was radiolabeled by a novel procedure which does not require the use of preiodinated Bolton-Hunter reagent (specific activities of 0.5-3.0 microCi/micrograms). Gel filtration chromatography of the radiolabeled preparation yielded two peaks. The early eluting peak contained disulfide stabilized aggregates with minimal interferon antiviral activity. The second peak contained activity that was consistently greater than or equal to that of the nonradiolabeled rIFN-gamma. Two bands with apparent molecular weights of 17,000 and 34,000 were observed when the second peak was analyzed by SDS gel electrophoresis. Fractions comprising each of the two chromatography peaks were pooled separately and subjected to gel filtration again on identical columns 24 h after completion of the first column run. The elution volumes of each peak remained unchanged suggesting that the two forms are not in rapid equilibrium. The plasma clearance rates of [125I]rIFN-gamma before and after purification by chromatography were initially rapid but multiphasic. The slower phases of clearance did not result from stable association of the rIFN-gamma with plasma proteins. In organ distribution studies, the liver and spleen sequestered significant amounts of [125I]rIFN-gamma; however, the highest concentration of rIFN-gamma was recovered in the kidneys. A functional nephrectomy procedure was used to further study the role of the kidneys in rIFN-gamma clearance. Eliminating the kidneys significantly increased the amount of rIFN-gamma retained in the circulation, particularly at later times when the vascular [125I]rIFN-gamma levels were approximately threefold higher than in nonnephrectomized mice.