The TCR couples antigen recognition and the transmission of activation signals. We report the expression of two TCR populations on the surface of T lymphocytes, one of which is linked to the cytoskeleton via the ζ chain. We also demonstrate that assembly of the CD3 subunits with cytoskeleton- associated ζ is necessary for their maximal localization to the cytoskeleton. The potential significance of these two receptor forms is underscored by differences observed in non-activated T cells; while detergent-soluble phosphorylated ζ appears as a 21-kDa protein, phosphorylated cytoskeleton-associated ζ appears as a 16-kDa form. This dichotomous phosphorylation pattern is rigidly maintained following activation, although each of the receptor populations undergoes different activation-dependent modifications: 1) levels of soluble phosphorylated 21- kDa ζ are enhanced, while phosphorylated 16-kDa cytoskeleton-associated ζ exhibits little change; 2) soluble non-phosphorylated 18-kDa ζ translocates to the cytoskeleton; 3) activation-dependent ubiquitinated ζ forms localize to both fractions, albeit with different kinetics. We also show that the protein tyrosine kinase Lck undergoes activation-dependent modifications end translocates to the cytoskeleton. The phosphorylation profiles of the dichotomous TCR populations in both non-activated and activated lymphocytes suggest that each population could regulate distinct cellular functions, possibly by select intermolecular associations.