Infection by a viable deletion mutant of BK virus (pm-522) of human embryonic kidney (HEK) cultures originating from different fetuses generated cell lines, designated pmHEK, resistant to superinfection by the virus. However, infection of HEK cultures by a cloned wild-type BK virus (wt-501) did not generate a cell line. In medium with 10% calf serum all pmHEK cells reached saturation densities significantly higher than those of HEK cells and could grow in medium containing 1% serum. They did not form colonies in soft-agar medium, and had limited life-spans greatly extended beyond that of HEK cells. These results suggest that pmHEK cells are partially transformed. T antigen was uniformly expressed by all pmHEK cells, while V antigen was present in only a small minority of the cells. Thirty to 5,000 copies of the viral DNA per cell were detected, primarily in a nonintegrated form, in all pmHEK cell lines and the clones isolated from one of them. Since at least in two pmHEK lines, the actual quantities of free viral DNA per cell were significantly greater than those estimated on the basis of the assumption that free viral DNA was produced only by the V-antigen-positive cells, it can be concluded that free viral DNA is also present in the V-antigen-negative cells predominant in the cell populations. Although the existence of a few copies of integrated viral sequences in pmHEK cells was not ruled out, the large amount of free viral DNA present in each cell probably plays a prominent role in the production of T antigen required for maintenance of transformation.