Epstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 10(5.9). We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4(+) T cells and CD16(+) CD56(+) NK cells. After virus challenge both CD4(+) T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16(+) CD69(+) cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4(+) T cells and CD16(+) NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4(+) T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4(+) T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4(+) T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.