The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if gammadelta+ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in gammadelta+ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in gammadelta+ IEL and in CD3+ cryptopatch cells, but not in CD3- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in gammadelta+ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR delta-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface gammadelta TCR. Our results are consistent with the hypothesis that a subset of gammadelta+ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal gammadelta+ T cell homeostasis still needs to be determined.