In this paper, we provide evidence that the vitronectin receptor (VNR) alpha v beta 3 is expressed on human NK cells. The presence of this VNR on freshly purified NK cells was demonstrated by flow cytometry analysis, as well as biochemically, after 125I-labeled surface lactoperoxidase labeling and immunoprecipitation. mAbs LM142 and LM609 specific for alpha v and alpha v beta 3, respectively, precipitated a heterodimer of alpha- and beta-chains with approximate molecular masses of 155 and 110 kDa under nonreducing conditions. Under reducing conditions, there was an apparent decrease in the molecular mass of the alpha-chain, which is likely to result from the release of a protein of 20 to 30 kDa linked by internal disulfide bond to the alpha v-chain. Integrin alpha v beta 3 expressed on NK cells became functional, i.e., was able to bind its ligand, vitronectin (VN), only after cellular activation or when costimulation with an additional signal was provided. Thus, NK cells adhered to plastic-immobilized VN only after IL-2 activation, and RGD-containing synthetic peptides or mAbs specific for alpha v beta 3 complex inhibited this binding. To assess the role of the VNR in signal transduction, anti-beta 3 mAb was used to cluster the VNR on NK cells and, thereby, mimic the process that occurs during formation of adhesive contacts. Cross-linking of VNR on fresh NK cells stimulated phosphorylation on tyrosine residues of several intracellular proteins. The major increase in tyrosine phosphorylation was observed in proteins of approximate molecular masses of 75 and 120 kDa. Therefore, signal transduction by the VNR on NK cells induced activation of intracellular protein kinases. Ligand engagement of the VNR on NK cells also costimulated cytokine production and proliferation of NK cells. Binding of NK cells to plastic-immobilized VN served as a costimulus with either anti-Fc gamma RIII or IL-2 to produce IFN-gamma, TNF-alpha, and cell proliferation. Our findings suggest that occupancy and subsequent clustering of VNRs play a role in the activation and function of human NK cells.