Recent work indicates that a novel homing receptor (HR)/endothelial ligand pair--the cutaneous lymphocyte-associated Ag (CLA) and E-selectin--is involved in targeting a unique skin-associated subpopulation of memory T cells to cutaneous sites of chronic inflammation. To investigate the regulatory mechanisms responsible for the generation of a memory T cell subset with skin-selective homing capability, we used multiparameter flow cytometry to assess the expression of CLA on T cells during initial T cell activation in secondary lymphoid tissues (the virgin to memory transition), and upon reactivation in skin. Our analyses indicate that in vivo induction of CLA (and E-selectin-binding ability) first occurs during the virgin to memory transition, and is regulated in a highly tissue-selective manner. The frequency of CLA expression on CD45RA+/RO+ transitional T cells within skin-associated peripheral lymph nodes was > fivefold higher than within the mucosal microenvironment of the appendix. In keeping with its role as a skin-selective homing receptor, levels of cell-surface CLA on T cells obtained from cutaneous blisters overlying delayed-type hypersensitivity sites were a mean 23-fold higher than on corresponding peripheral blood T cells, including all time points of lesional evolution. However, comparisons of CLA expression on resting vs activated skin blister T cells (HLA-DR - vs +, respectively) indicated that CLA expression, which is already quite high on the resting subset, appeared to be further up-regulated upon activation in skin. The critical involvement of local microenvironments in the regulation of CLA expression was also supported by in vitro studies demonstrating the necessity of specific secondary signals for the induction of CLA glycoproteins on mitogen-activated T cells. CLA up-regulation on both the virgin and memory T cell subsets was dependent on the presence of TGF-beta 1 (or, to a lesser extent, IL-6), but not a wide variety of other cytokines. Thus, the development of the CLA+ memory T cell subset is likely a product of the cumulative experience of those T cells with respect to local microenvironments at previous sites of activation, perhaps involving differential availability of bioactive TGF-beta 1 and/or IL-6 (both cytokines produced in skin). Repeated activation in skin or skin-associated peripheral lymph nodes may act to reinforce CLA expression on T cells functionally-associated with skin, and thus enhance the functional efficiency of these cells by preferentially focusing their recirculation to the skin or related sites.