The function of the CD44 glycoprotein as an adhesion molecule was directly tested by transformation of a CD44 cDNA into mouse fibroblasts. This cDNA was expressed as a heavily modified cell surface protein reactive with monoclonal antibodies that recognize glycoproteins now identified in primates as CD44. Independent transfectants exhibited a new self-adhesive phenotype, forming large aggregates when placed in suspension. In variants derived from a clone of cells, aggregation competence segregated with expression of the transfected gene. This CD44-mediated adhesion was blocked specifically by monoclonal antibodies binding one immunologically defined region of CD44. Nontransfected L cells did not self-aggregate but were capable of adhering to the transfectants, indicating that at least one ligand for this adhesion molecule is expressed by mouse fibroblasts.