Biotinylation and radioiodination have been compared for labeling lymphocyte-surface proteins and the labeled proteins symmetrically immunoprecipitated with antibodies recognizing major lymphocyte markers such as the murine Thy-1, CD25 (the alpha subunit of the interleukin-2 receptor), CD45, and human CD2 glycoproteins. The detection of biotinylated proteins by enhanced chemiluminescence after transfer to nitrocellulose was found to be fast and as efficient as the detection of iodinated proteins by autoradiography. The vectoriality of cell-surface biotinylation was ascertained by two-dimensional electrophoresis of the cellular extract in which the major cytoplasmic proteins were not found biotinylated. This nonradioactive labeling procedure offers a convenient and efficient alternative to radiolabeling of cell surfaces for the biochemical analysis of extracellular domains of membrane proteins.