Two populations of CD8(+) IEL generally express restricted, but apparently random and non-overlapping TCR repertoires. Previous studies in mice suggested that this could be explained by a dual origin of CD8(+) IEL, i.e. that CD8alphabeta(+) IEL derive from a few peripheral CD8(+) T cell lymphoblasts stimulated by microbial antigens in gut-associated lymphoid tissue, whereas CD8alphaalpha(+) IEL descend from an inefficient intestinal maturation pathway. We show here that the gut mucosa, instead, becomes seeded with surprisingly broad and generally non-overlapping CD8 IEL repertoires and that oligoclonality is induced locally after microbial colonization. In germ-free (GF) rats, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed surprisingly diverse TCR Vbeta repertoires, although beta-chain diversity tended to be somewhat restricted in the CD8alphaalpha(+) subset. CDR3 length displays in individual Vbeta-Cbeta and Vbeta-Jbeta combinations generally revealed polyclonal distributions over 6-11 different lengths, similar to CD8(+) lymph node T cells, and CDR3beta sequencing provided further documentation of repertoire diversity. By contrast, in ex-GF rats colonized with normal commensal microflora, both CD8alphabeta(+) and CD8alphaalpha(+) IEL displayed oligoclonal CDR3 length distributions for most of the Vbeta genes analyzed. Our data suggest that microbial colonization induces apparently random clonal expansions of CD8alphabeta(+) and CD8alphaalpha(+) IEL locally in the gut.