Placenta growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in pathological conditions. PlGF exerts its biological function through the binding and activation of the seven immunoglobulin-like domain receptor Flt-1, also known as VEGFR-1. Here, we report the first detailed mutagenesis studies that provide a basis for understanding molecular recognition between PlGF-1 and Flt-1, highlighting some of the residues that are critical for receptor recognition. Mutagenesis analysis, performed on the basis of a structural model of interaction between PlGF and the minimal binding domain of Flt-1, has led to the identification of several PlGF-1 residues involved in Flt-1 recognition. The two negatively charged residues, Asp-72 and Glu-73, located in the beta3-beta4 loop, are critical for Flt-1 binding. Other mutations, which bring about a significant decrease in PlGF binding activity, are Gln-27, located in the N-terminal alpha-helix, and Pro-98 and Tyr-100 on the beta6 strand. The mutation of one of the two glycosylated residues of PlGF, Asn-84, generates a PlGF variant with reduced binding activity. This indicates that, unlike in VEGF, glycosylation plays an important role in Flt-1 binding. The double mutation of residues Asp-72 and Glu-73 generates a PlGF variant unable to bind and activate the receptor molecules on the cell surface. This variant failed to induce in vitro capillary-like tube formation of primary endothelial cells or neo-angiogenesis in an in vivo chorioallantoic membrane assay.