Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer's patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3epsilon, TCRzeta, TCRgamma, and TCRbeta and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRbeta repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRbeta transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRbeta repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vbeta4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRbeta repertoire compared with the spleen and pronephros TCRbeta repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified alphabeta TauCR repertoire is maintained in fish IELs in the absence of Peyer's patches and mesenteric lymph nodes, whereas the restricted diversity of mouse alphabeta IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.