Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.