We investigated the characteristics of lymphocytes propagated from biopsies of the mesenteric lymph nodes, liver, and ileum of a human multivisceral allograft in order to provide functional evidence for the presence or absence of rejection and graft-versus-host disease (GVHD). The recipient was a 39-month-old girl with secretory diarrhea due to microvillus inclusion disease and end-stage liver disease secondary to prolonged parenteral nutrition. She developed a multifocal posttransplant lymphoproliferative disorder (PTLD) and died 37 days after transplantation. Four pairs of sequential mesenteric lymph node and liver biopsies (13, 17, 24, and 33 d posttransplant) and a single ileal biopsy (31 d posttransplant) were placed in culture with recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). T-cell phenotyping of cultured cells showed that CD8+ cells became dominant in all three tissues. The alloreactivity of biopsy-grown cells was determined using the primed lymphocyte test (PLT) and cell-mediated lympholysis test (CML). The proliferative and/or cytolytic responses of biopsy-grown cells to donor but not recipient or third party cells provided evidence for rejection and absence of GVHD. This donor-specific alloreactivity was detected before there was histologic evidence of rejection and during the period of active lymphoproliferation. This study suggests that the functional characterization of graft-infiltrating lymphocytes is useful in defining the immunologic events following multivisceral transplantation.