Leucine chloromethylketone labeling of viable S37 cells was preferential for the plasma membrane fraction. The pattern of radiolabeling of the plasma membrane proteins was time dependent. After 5 min the radiolabel was localized with glutamyl transpeptidase, and subsequently with other physiologically active proteins as a function of time after incubation. Labeling of proteins was temperature dependent and incubation of viable S37 cells with the radiolabeled substrate at 0 degrees C yielded little or no radioactivity localized in the plasma membrane. The molecular weight of one radiolabeled substrate--membrane protein complex was estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis to be between 100,000-200,000.