To study the self-reactivity of rat lymphocytes as a function of age we applied an in vitro correlate of the experimental autoimmune orchitis (EAO). Previous analyses indicated that in this in vitro model, T lymphocytes react specifically against autoantigens when cocultured with syngeneic testis cells. The EAO response shows tissue specificity and is determined by the MHC. The self-reaction is measured in a cytostasis microassay, which is based on estimating growth inhibition of testis cell-derived monolayer formation in a terminal 51Cr uptake assay. We compared the self-reactivity of individual rats of varying ages (3 to 26 months) by testing their in vitro EAO responsiveness. In parallel, their reactivity to alloantigens and a polyclonal T lymphocyte activator (Con A) was monitored. We found a decrease in the allo- and the mitogen responsiveness in aged animals (from 20 months on) compared to young controls (3 months). The self-reactivity, in contrast, was in all cases increased. The increased EAO response was found not to be due to age-dependent alteration of the relevant autoantigens nor to changed functions of accessory non-T cells. The enhanced autoimmune reactivity was rather a property of the T cell compartment. We believe that these results indicate an age-dependent loss of T lymphocyte self-tolerance, which may play an active role in the pathogenesis of autoimmune diseases in the aging organism.