We have developed a clonal transformation assay for Epstein-Barr virus which uses adult human leukocytes as target cells. The target cells were isolated from Epstein-Barr seronegative donors, and the same donor's cells could be studied repeatedly over long periods of time. When these cells were transformed by Epstein-Barr virus and had proliferated sufficiently to be studied, they had an average cloning efficiency of 3%. Assuming this average cloning efficiency obtains at the onset of transformation, we calculate that transformation by Epstein-Barr virus leads to immortalization maximally of about 1 in 30 of the adult peripheral leukocytes exposed to the virus. Studying the number of colonies transformed as a function of the amount of virus to which the cells are exposed indicates that a single DNA-containing virus particle is sufficient to transform a cell. All of the transformed clones studied harbored viral DNA. This technique will now permit, for the first time, our studying clonal variations in adult peripheral leukocytes transformed by Epstein-Barr virus as a function of input multiplicity of the virus and of the donor's immune status.