Thymic rosettes, structures consisting of 3-30 thymic lymphoid cells attached to a central macrophage or dendritic cell, were released from mouse thymus tissue by collagenase digestion. They were shown to be preexistent structures within the thymus, but to be subject to extensive exchange with free thymocytes under certain conditions. An isolation procedure was developed, using a new technique of zonal unit-gravity elutriation, which minimized exchange and produced a completely pure sample of the larger rosettes. The rosette-associated thymocytes were analyzed by two- and three-color immunofluorescent staining and flow cytometry. The dominant cell type was a small, CD4+CD8+, cortical-type thymocyte. However, all of the established thymus subpopulations defined by CD4 and CD8, including CD4-CD8+ and CD4+CD8- mature thymocytes and CD4-CD8- early thymocytes, were also present in rosettes. Very few of the cells present were of an intermediate or transitional phenotype. Rosette-associated thymocytes were somewhat enriched in large dividing thymocytes, in CD4-CD8- thymocytes, and in mature thymocytes expressing the T-cell antigen receptor-CD3 complex. Their most striking characteristic was a marked depletion in small thymocytes lacking surface H-2K expression, a major population among free thymocytes. The physiological role of the rosette structure is discussed, and it is suggested that the heterogeneity of the associated thymocytes in part reflects the existence of different types of rosettes in different areas of the thymus.