A subpopulation of human peripheral blood natural killer (NK) cells, defined by sedimentation at Percoll high buoyant densities (P greater than 1.0635-1.0640 g/ml) and unresponsiveness to interleukin-2 (IL-2), contained two distinct populations based on the intensity of CD16 (FcR gamma III) expression, namely CD16dim and CD16bright. This resting subpopulation of NK cells differed from the total population of peripheral blood NK cells, by containing a larger proportion of CD16dim cells, by the total absence of CD56bright CD16- cells, and by an inability to respond to high concentrations (500 U/ml) of rIL-2 despite the expression of an intermediate affinity (p70) IL-2R. Both CD16dim and CD16bright NK cells expressing high affinity IL-2R were initially generated following co-culture of resting NK cells with gamma-irradiated MM-170 cells and IL-2, but CD16bright NK cells became the dominant cell type later in culture. The CD16 molecule was not involved in the differentiation of resting NK cells since solid-phase-bound anti-CD16 monoclonal antibody neither enhanced nor inhibited NK cell generation. These studies demonstrate that the resting subpopulation of peripheral blood NK cells expresses a unique CD16 profile, that CD16 expression increases during NK cell generation, and that CD16 is not involved in the differentiation process.