Jacalin is a multimeric plant lectin able to interact with the lymphocyte cell-surface molecule CD4, a known receptor for the human immunodeficiency virus type 1 (HIV-1). Moreover, jacalin is able to block HIV-1 infection of CD4+ lymphoblastoid cells. Here we studied whether jacalin prevents HIV-1 gp120-CD4 interactions. We found (i) that jacalin did not inhibit HIV-1 Lai-induced syncytium formation that requires gp120-CD4 interactions; (ii) that jacalin prevented neither rgp120 binding to cell-surface CD4 nor sCD4 binding to viral envelope proteins expressed at the surface of HIV-1-infected lymphoblastoid cells; (iii) that jacalin did not compete for binding to CD4 with anti-CD4 mAb specific for the CDR2- or CDR3-like regions of the D1 domain of CD4; (iv) that jacalin did not bind a recombinant soluble molecule containing the D1/D2 domains of CD4; and, (iv) that jacalin binding to CD4 is inhibited by sugars known to interact with the lectinic-site of jacalin. These data have implications for the understanding of the mechanism by which jacalin blocks HIV-1 infection of CD4+ cells.