Here we examined the changes in NADPH diaphorase (NADPHd) and inducible nitric oxide synthetase (iNOS) positivity in the medulla of the mouse thymus in response to treatment with the superantigen, Staphylococcal enterotoxin B (SEB). A few NADPHd+ and iNOS+ cells scattered in the medulla were detected in the thymi of naive mice. SEB induced the appearance of a large number of NADPHd+- and iNOS-immunoreactive cells in the thymic medulla. In the thymus of iNOS-deficient mice, a total absence of these NADPHd+ and iNOS+ medullary cells was found both under basal conditions and after SEB stimulation. With the NADPHd reaction, only endothelial staining was detected in the thymi of iNOS-deficient mice. Our data indicate that NADPHd+ cells in the thymic medulla express iNOS and that SEB induces iNOS expression in the mouse thymus.