The Spodoptera exigua galactose binding lectin, extracted from hemolymph by affinity chromatography, was comprised of large molecular weight aggregates (100-700 kDa). SDS-PAGE of this lectin preparation revealed it to contain 2 subunits of 33.2 and 34.4 kDa in equimolar concentrations. These subunits had similar amino acid profiles, possessed identical N-terminal sequences and reacted equally to a bank of antilectin monoclonal antibodies. By staining Western blots with various lectin conjugate probes, we demonstrated that the 34.4 kDa subunit contains complex mannose residues, suggesting that this subunit is the glycosylated form of the 33.2 kDa subunit. The N-terminal sequence of the S. exigua lectin was distinct from other invertebrate galactose binding lectins. Light microscopy in combination with immunoelectron microscopy was used to localize the S. exigua lectin in the granules of the granulocyte class of hemocytes. Degranulation of these cells resulted in the release of the lectin. Isotope incorporation studies followed by immunoprecipitation with a S. exigua monoclonal antibody demonstrated that the fat body was the major site of lectin synthesis. Similar studies with hemocyte monolayers did not result in the production of detectable levels of 35S-labeled S. exigua lectin.