Using an indirect fluorescent antibody technique with frozen sections, the localization of thymosin β9 was investigated for the first time in bovine thymus, spleen, lung, muscle and liver. The antibodies used have been raised against the N-terminal fragment 1–14 of thymosin β9 in order to minimize the cross-reactivity with thymosin β4 which was found to be also present in bovine tissues. The specific antibodies against thymosin β9 raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin β4 in different tissues. Although thymosin β9 was first isolated from calf thymus, it could be also detected in other bovine organs. The highest density of positive immunoreaction was found to be in spleen sections. In the muscle tissue a pronounced fluorescence intensity was present in the region of the sarcolemn.