Autoimmune diseases develop in selected normal mouse strains when thymectomy (Tx) is performed at 3 days of age (d3-Tx). Insufficient T cell regulation after Tx may result from a defect in regulatory T (Treg) cells or from an augmented effector T (Teff) cell number/pathogenicity. We have previously shown that Tx at 3 wk (wk3-Tx), the age of massive islet Ag release, accelerates diabetes onset. We now have determined diabetes incidence in d3-Tx nonobese diabetic mice and compared the frequency and function of their Teff and Treg cells with those of wk3-Tx mice. We found that d3-Tx had no effect on diabetes incidence, but induced gastritis. After day 3 and week 3 Tx, Treg cells were fully competent and their frequency increased. The number of diabetogenic T cells was greatly amplified after wk3-Tx and likely overcame Treg cell control, leading to an early tolerance breakdown. By contrast, in d3-Tx mice, activation concerned few cells and Teff cell amplification remained controlled. This suggests that Tx enhances autoimmunity when it coincides with the first encounter of autoreactive T cells with their cognate Ag. The relationship between Tx-induced lymphopenia, tissue remodeling, and autoimmunity is discussed.