FcgRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific anti-bodies (bsAbs) targeting FcgRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcgRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgRIIIB extra-cellular domains. These sdAbs bind FcgRIIIA 1 NK cells and FcgRIIIB 1 poly-morphonuclear cells, but not FcgRI 1 or FcgRII 1 cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcgRIII with a K D in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-g production, respectively. These anti-FcgRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgRIII killer cells to target and destroy tumor cells.