Donor-specific, alloreactive T cell lines may be grown from cells infiltrating human renal allografts. These T cell lines utilize restricted T cell receptor (TCR) β-chain variable (Vβ) gene repertoires, although long-term culture appears to be necessary for restriction to be observed. This study was undertaken to determine the effects of potential selective pressures on the TCR repertoires of allograft-infiltrating cells. TCR Vβ repertoires of 30 allograft-derived T cell populations, cultured for defined, short time periods, were examined using polymerase chain reaction. When first derived, Vβ repertoires of graft-infiltrating T cells were as heterogeneous as those of peripheral blood lymphocytes (PBL). There was no relationship between the length of time an allograft was in situ or the extent of HLA mismatch and repertoire heterogeneity. Repertoire restriction was positively correlated with the length of time cells were cultured in vitro. Long-term, alloreactive mixed lymphocyte reactions (MLR), established from normal, unsensitized PBL, also demonstrated Vβ repertoire restriction during expansion in vitro. Restricted alloreactive populations emerged much more slowly from the MLR than from the allograft-derived cultures, however, implying that graft infiltrates contain previously activated populations of T cells. This observation, taken together with the fact that long-term, graft-derived cell lines maintain donor specificity, suggests that functional subsets must be allowed to emerge from heterogeneous infiltrates before TCR repertoire may be correlated with alloreactivity and/or graft rejection.