Thy-1+ lymphocytes, detectable by quantitative serum absorption, arise in cultures of spleen cells from congenitally athymic (nu/nu) mice supplemented with supernatants from cultures of normal spleen cells stimulated with the T cell mitogen concanavalin A. Pretreatment of nu/nu spleen cells with appropriate anti-Thy-1 alloantibody and complement prior to culture reduces their capacity to generate Thy-1+ cells by about 90%. This shows that the majority of cells proliferating in these cultures are descendants of Thy-1+ cells which can be detected in the original nu/nu spleens. Experiments aimed at exploring the function of these Thy-1+ cells after culture in conditioned medium revealed that within one or two days after culture initiation, strong cooperative activity for a humoral response to xenogeneic erythrocytes can be detected. In mixtures of bone marrow-derived lymphocytes from various mouse strains with cultured nu/nu spleen cells, it was observed that T-B cooperation is not H-2-restricted. Attempts at inducing T cell-mediated cytotoxicity to alloantigens in such cultures of nu/nu spleen cells were unsuccessful. In contrast, nonspecific cytotoxicity which was attributed to natural killer cells was regularly observed and could be maintained in these cultures over extended periods.