The present study shows that a mAb (H4C4) developed against human peripheral blood adherent cells has the unusual property of inducing in vitro homotypic aggregation of several types of hemopoietic cells and cell lines. The Ag recognized by mAb H4C4 is a 85-kDa glycoprotein that corresponds to the human Ag CD44 (equivalent to murine Pgp-1), as determined by protein purification, immunologic cross-reactivity studies, and tryptic fragment sequencing. In addition to H4C4, other mAb directed against some, but not all, epitopes of CD44(Pgp-1) were capable of inducing cell aggregation. This process was temperature sensitive and was almost totally abrogated by cytochalasin B but was unaffected by sodium azide, colchicine, EGTA, trifluoperazine, or staurosporin. A role for CD44 (Pgp-1) in cell-to-cell adhesion was further indicated by an inverse relationship observed between spontaneous aggregation of some hemopoietic cell lines and cell-surface expression of CD44(Pgp-1). These observations provide evidence for a fundamental role of CD44(Pgp-1) in cellular aggregation phenomena with an involvement of the cytoskeleton.