Bis(tri-n-butyltin)oxide (TBTO) is one of the organotin compounds that have been used as biocides and occur as persistent environmental pollutants. Human exposure to these compounds occurs through consumption of meat and fish products in which they accumulate. The most sensitive endpoint of TBTO exposure is immunotoxicity. TBTO causes thymus atrophy and thereby interferes with T-lymphocyte-mediated immune responses. Tributyltin compounds have been found to adversely affect a wide range of cellular components and processes in many species, organ systems, and cell types. Both inhibition of proliferation and induction of apoptosis have been observed in thymocytes. We conducted microarray experiments in mice and rats in order to investigate if the immunosuppressive actions of TBTO could be detected by gene expression profiling, and if so, to elucidate the mechanisms of action. Gene expression changes that were detected in mouse thymuses after exposure to a maximum tolerable dose of TBTO correlated to previously observed effects. Most notably, reduction of expression of cell surface determinants and T-cell receptor chains, suppression of cell proliferation, and a possible involvement of nuclear receptors in interference with lipid metabolism by TBTO were observed. The TBTO-induced thymus involution may therefore primarily be caused by inhibition of thymocyte proliferation. In contrast, in rats only limited effects of a lower dose of TBTO were found at the gene expression level in the thymus, even though thymus involution was observed. Here, most gene expression regulation by TBTO was detected in the liver. These preliminary results indicate that gene expression analysis is able to reveal effects of TBTO and to gain insight into its molecular mechanism of action. It may even be a suitable tool to investigate immunotoxicology in general. However, dose and inter-species differences are apparently clearly reflected in the gene expression profiles.