A lymphoproliferative assay was developed to study cell-mediated immunity (CMI) to BK virus (BKV), a human papovavirus, in healthy volunteer subjects. Responses to ultraviolet-inactivated antigen prepared from BKV-infected fibroblasts were compared to those elicited against a mock antigen preparation and an unrelated control antigen (tetanus toxoid, TET). CMI to BKV and TET were contrasted with humoral immunity as measured by enzyme-linked immunosorbent assay (ELISA). Specificity of the assay was confirmed by absence of response to mock antigen in all subjects studied. Positive response to BKV antigen was observed in all of 15 seropositive individuals but not in 5 neonates or 1 seronegative child. Similarly, all TET seropositive (n = 13) but no seronegative subjects (n = 2) responded to TET. The magnitude of lymphoproliferation to either antigen did not correlate with antibody titer. Additionally, the frequency of peripheral blood BKV-specific proliferating lymphocytes was determined by limiting dilution analysis (LDA). The frequency was approximately tenfold less than that observed for TET in the same group of subjects (1/30,300 vs 1/2,700). This may be due to differences in route and frequency of antigen exposure, both of which are unknown, at present, for BKV.