The aim of this study was to examine the effect of cytokines on different subsets of NK cells, while especially focusing on CD16⁻ CD56^dim cells and CD16⁻ CD56^bright cells. When human peripheral blood mononuclear cells (PBMC) were cultured with a combination of IL‐2, IL‐12 and IL‐15 for several days, a minor population of CD56^bright NK cells expanded up to 15%, and also showed potent cytotoxicities against various cancer cells. Sorting experiments revealed that unconventional CD16⁻ CD56⁺ NK cells (CD16⁻ CD56^dim NK cells and CD16⁻ CD56^bright NK cells, both of which are less than 1% in PBMC) much more vigorously proliferated after cytokine stimulation, whereas predominant CD16⁺ CD56^dim NK cells proliferated poorly. In addition, many of the resting CD16⁻ CD56^bright NK cells developed into CD16⁺ CD56^bright NK cells, and CD16⁻ CD56^dim NK cells developed into CD16⁻ CD56^bright NK cells and also further into CD16⁺ CD56^bright NK cells by the cytokines. CSFE label experiments further substantiated the proliferation capacity of each subset and the developmental process of CD16⁺ CD56^bright NK cells. Both CD16⁻ CD56^dim NK cells and CD16⁻ CD56^bright NK cells produced large amounts of IFN‐γ and Fas‐ligands. The CD16⁺ CD56^bright NK cells showed strong cytotoxicities against not only MHC class I (−) but also MHC class I (+) tumours regardless of their expression of CD94/NKG2A presumably because they expressed NKG2D as well as natural cytotoxicity receptors. The proliferation of CD16⁺ CD56^bright NK cells was also induced when PBMC were stimulated with penicillin‐treated Streptococcus pyogenes, thus suggesting their role in tumour immunity and bacterial infections.