We have described previously an adhesive protein on neutrophils (PMN) which recognizes fibrinogen, fibronectin (Fn), von Willebrandt's factor, vitronectin, collagen, and synthetic peptides containing the Arg-Gly-Asp (RGD) sequence (Gresham et al., J. Cell Biol. 108, 1935-1943, 1989). We have called this oligospecific receptor the leukocyte response integrin (LRI). Engagement of LRI leads to both increased ingestion via PMN IgG Fc receptors and to adhesion and chemotaxis to certain extracellular matrix proteins. Now, we have purified an RGD-binding receptor from DMSO-differentiated HL-60 cells (dHL-60) by peptide affinity chromatography which has the biochemical, immunologic, and functional characteristics of LRI. The purified protein contains two bands of 135 and 90 kDa under nonreducing conditions SDS-PAGE. Immunologic characterization of the dHL-60 RGD receptor showed that, by Western blot and ELISA, the lower M(r) band was recognized by mAb 7G2, raised against placental beta 3, which is known to inhibit LRI function. However, despite this functional and immunologic cross-reactivity with beta 3, the receptor was not recognized efficiently by a polyclonal antibody to placental RGD-binding proteins, predominantly alpha v beta 3. Moreover, polyclonal antibody raised to the dHL-60 receptor (Ab1) did not react with placental RGD-binding proteins. By immunoprecipitation or ELISA, we demonstrated that the purified RGD-binding receptor was not alpha IIb beta 3 or alpha v beta 3 and did not contain the integrin chains alpha 4, beta 2, or beta 7. Functionally, Ab1 totally inhibited Fn-stimulated ingestion by PMN. Moreover, Ab1 inhibited phagocytosis stimulated by the peptide KGAGDV, which is the most specific ligand for LRI currently known, and Ab1 inhibited the binding of KGAGDV-coated microspheres to PMN and monocytes. FACS analysis with Ab1 showed staining of monocytes, PMN, and lymphocytes but not platelets or erythrocytes. We conclude that LRI is a novel RGD-binding receptor which exists on leukocytes and which shares an antigenic epitope(s) with beta 3. This receptor recognizes multiple RGD-containing ligands and can mediate signal transduction for adhesion, chemotaxis, and activation of increased phagocytic potential by PMN and monocytes.