To correlate the histologically observed thymic abnormalities with the cellular immunodeficiency found in Down's syndrome (DS), thymus fragments and thymocyte suspensions from 14 noninstitutionalized DS subjects were studied. Histologic examination and immunohistologic studies using an anticluster of differentiation (CD) 1 monoclonal antibody showed a contracted cortex due to cortical thymocyte depletion. When DS unselected thymocytes were phenotyped, a significant reduction of CD3-, CD1-, CD4-, and CD8-positive cells was found as compared to controls. To evaluate if the deficient expression of these markers was due to the reduction of thymocyte subsets identifiable on the basis of their physical properties, we separated DS unselected thymocytes into 10 fractions by continuous Percoll density gradient centrifugation. DS thymuses were almost completely devoid of high density thymocytes. Since in normal thymus, these cells correspond to small CD1+, CD4+, CD8+, and 50% CD3+ cortical thymocytes, their absence may explain the unrestricted reduction of markers on DS unfractionated thymocytes. Furthermore DS thymuses appeared to be enriched in CD1+ first fraction (Fr1) low density thymocytes of the Percoll gradient. Fr1 CD1+ cells constitute the main spontaneously proliferating pool in normal human thymus. When the spontaneous proliferating activity of DS Fr1 was compared to that of the control, a significant reduction was observed. This reduction associated with the absence of high density thymocytes, with the reduction of cells expressing alpha- and beta-chains of the T cell receptor and in conclusion with the lymphocyte depletion, suggests that in DS thymuses there is a deficient expansion of immature T cells resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool able to differentiate into functionally mature T cells.