In this study, we compared the natural killer (NK) cells that lyse HSV-1-infected NK(HSV-1) or uninfected [NK-(FS)] fibroblasts to those that lyse K562 erythroleukemia cells [NK(K562)]. Activity against all three targets was found in Percoll gradient fractions enriched for large granular lymphocytes, which suggests that these effector cells have a common morphology. In competition studies between 51Cr-labeled targets and unlabeled targets, both the infected and the uninfected fibroblasts competed for lysis of NK(HSV-1) and NK(FS) activity, whereas K562 cells competed poorly. In contrast, when 51Cr-labeled K562 cells were used, the unlabeled K562 cells competed well, but HSV-1-infected and uninfected fibroblasts competed poorly. Panning studies and complement elimination experiments using monoclonal antibodies were performed to describe cell surface markers on the NK cell populations. Treatment with an antibody to an la framework antigen reduced NK(HSV-1) but not NK(K562) activity. In contrast, the majority of NK(K562) effectors were recognized by antibodies to the E-rosette receptor (Lyt-3 and OKT11A), whereas NK(HSV-1) activity was much less sensitive to this antibody. OKM1, OKT10, and Leu-7 (HNK-1) markers were found on a portion, but not all, of the cells that lysed both the HSV-FS and K562 targets, while treatment with HLA plus complement totally abrogated both NK activities. Taken together, these data are consistent with the concept that human NK cells are heterogeneous and that we are dealing with at least three subpopulations of effector cells--one that kills the infected or uninfected fibroblasts; one that kills K562 cells; and a third population that may be able to kill all three targets. Patient studies provide additional evidence for heterogeneity within the NK cells that lyse the fibroblasts and K562 cells. We have studied a number of individuals who have normal NK activity with one target (HSV-FS or K562) but have low or no activity against the other. These patients provide strong evidence not only that NK cells are heterogeneous but also that these NK subpopulations can be regulated independently of each other in vivo.