Cultured human thymic tissue has been transplanted into many patients with T cell dysfunction; however, little is known about the effect of in vitro culture on thymic tissue. Human postnatal thymic organ cultures were established in vitro to study the growth potential of the thymic epithelium and the expression of intracellular and surface antigens with time in culture. Marked depletion of bone marrow-derived cells was observed within 3 weeks of initiation of organ cultures although some viable CD3+ cells could still be detected. Thymic epithelial cells in in vitro explants continued to express MHC class I and class II antigens as well as cytokeratins. Thymic epithelial cells within cultured thymic organ slices maintained their postnatal growth potential, in that cytokeratin-positive epithelial monolayers could be established in vitro from these thymic slices up to 12 weeks after initiation of organ culture. Thus, thymic explants remained viable in culture and could potentially be used to reconstitute immunity in T cell deficient patients.