A sensitive radioimmunoassay for eucaryotic elongation factor Tu (eEF-TU) was developed using radioiodinated elongation factor T (eEF-T) and goat anti-(rabbit eEF-T) immunoglobulins coupled to a solid support. eEF-T was iodinated with 125I to a specific activity of 7 x 10(3) counts min-1 ng-1 using a system employing lactoperoxidase and glucose oxidase coupled to a solid support. The assay exhibits a limit of detection of about 1 ng eEF-TU and an intraassay variability of < 10%. By using the radioimmunoassay, it was found that eEF-Tu is a major non-hemoglobin protein of rabbit reticulocyte postribosomal supernatant proteins, comprising about 3% of the total hemoglobin and 10--13% of the non-hemoglobin proteins. Similar results were found for a number of different tissues and cells, including rabbit heart, brain, liver and kidneys, as well as both induced and non induced Friend erythroleukemia cells. Values of eEF-Tu ranged from 1% of supernatant proteins in heart to about 11% in noninduced erythroleukemic cells. The levels of eEF-Tu in these mammalian tissues were comparable to the level of the homologous factor EF-Tu in Escherichia coli. It has previously been found that EF-Tu constitutes about 6--8% of the supernatant proteins of E. coli [Furano, A. V. (1975) Proc. Natl Acad. Sci. USA, 72, 4780--4784]. The level of eEF-Tu in reticulocytes was compared to the abundance of other components of protein synthesis in reticulocytes, such as translocase (eEF-G), tRNA, ribosomes and eIF-2. In all cases eEF-Tu was present in large excess over these other components.