The major surface glycoprotein of Leishmania chagasi promastigotes showed crossreactivity with fibronectin (Fn), a large glycoprotein that is a major constituent of the extracellular matrix of most mononuclear cells. Polyclonal and monoclonal antibodies against Fn precipitated two molecules of 63-58 kDa from the lysates of both 125I and [35S]methionine-labeled promastigotes. In addition, a monoclonal antibody against a 15-kDa fragment of Fn containing the Arg-Gly-Asp-Ser (RGDS) sequence and several polyclonal monospecific mouse antibodies against a synthetic RGDS peptide also recognized the above two molecules. The attachment of Leishmania promastigotes to mouse peritoneal macrophages in vitro was partially inhibited when promastigotes were treated with F(ab')2 fragment of an anti-Fn IgG. Identical results were obtained by saturating the Fn receptors on macrophages using different peptides containing the RGDS sequence. Moreover, antigen preparations rich in glycoprotein 63 could efficiently promote the attachment and spreading of 3T3 mouse fibroblasts to surfaces coated with the antigen. These results clearly suggest that the gp63 of L. chagasi promastigotes is an Fn-like molecule that shares certain biological and molecular characteristics with Fn.