The application of genomics-based technology in toxicological research may finally provide better tools for chemical safety testing since a wealth of information can be obtained about the mode of action of toxic compounds. Furthermore, by using this technology signature gene expression profiles may be identified for the classification of compounds. The overall aim of our work is to generate in vitro gene expression fingerprints that are predictive for direct toxicity to the immune system. In the present study the biocide and environmental pollutant bis(tri-n)butyltin)oxide (TBTO) was used as a model immunotoxicant. TBTO is known to cause thymus atrophy in rodents and thereby interferes with T lymphocyte-mediated immune responses. To study the in vitro effect of TBTO on gene expression we exposed primary thymocytes of C57Bl/6 mice for 3, 6 and 11 h to various non-cytotoxic concentrations of this immunotoxicant, i.e. concentrations that resulted in less then 20% decrease in viability. The experiments were performed using Agilent whole mouse genome (44 K) oligonucleotide microarrays. Upon multivariate analysis, data were further studied using gene ontology term enrichment and pathway finding tools. Pathways and processes found to be affected by TBTO include lipid metabolism, apoptosis, cell cycle control, glucocorticoid receptor signaling, and regulation of transcription. The extent to which these processes were affected, was different among the exposure conditions. Stimulation of glucocorticoid receptor signaling appeared to be a relevant mechanism of action, irrespective of the condition used. In short, the results of our study showed that genes involved in a number of physiologically relevant processes were differentially expressed upon exposure of primary thymocytes to TBTO.