BACKGROUND Cytomegalovirus (CMV) infection remains a major complication after kidney transplantation. Baseline CMV risk is typically determined by the serological presence of preformed CMV-specific immunoglobulin (Ig) G antibodies, even though T-cell responses to major viral antigens are crucial when controlling viral replication. Some IgG-seronegative patients who receive an IgG-seropositive allograft do not develop CMV infection despite not receiving prophylaxis. We hypothesized that a more precise evaluation of pretransplant CMV-specific immune-sensitization using the B and T-cell enzyme-linked immunospot assays may identify CMV-sensitized individuals more accurately, regardless of serological evidence of CMV-specific IgG titers. METHODS We compared the presence of preformed CMV-specific memory B and T cells in kidney transplant recipients between 43 CMV IgG-seronegative (sR(-)) and 86 CMV IgG-seropositive (sR(+)) patients. Clinical outcome was evaluated in both groups. RESULTS All sR(+) patients showed a wide range of CMV-specific memory T- and B-cell responses. High memory T- and B-cell frequencies were also clearly detected in 30% of sR(-) patients, and those with high CMV-specific T-cell frequencies had a significantly lower incidence of late CMV infection after prophylactic therapy. Receiver operating characteristic curve analysis for predicting CMV viremia and disease showed a high area under the receiver operating characteristic curve (>0.8), which translated into a high sensitivity and negative predictive value of the test. CONCLUSIONS Assessment of CMV-specific memory T- and B-cell responses before kidney transplantation among sR(-) recipients may help identify immunized individuals more precisely, being ultimately at lower risk for CMV infection.