Human Siglec-5 is a sialic acid binding immunoglobulin (Ig)-like lectin (Siglec), comprising one N-terminal IgV-SET domain followed by three IgC2-SET domains, and a cytoplasmic domain with ITIM and SAP motifs which regulate cell signalling. We report the differential distribution of hSiglec-5 on neutrophil and macrophage subsets in tissues using monoclonal antibodies, 1A5 and 2H8, which require the first IgC2-SET domain for binding. Interestingly, hSiglec-5 was especially prominent on macrophages in reactive lymph nodes. We have identified four isoforms of hSiglec-5 possessing three (hSiglec-5-3L and -3C) or four (hSiglec-5-4L and -4S) extracellular domains linked to long (hSiglec-5-3L and -4L) or short (hSiglec-5-4S) cytoplasmic tails or existing as a soluble isoform (hSiglec-5-3C). hSiglec-5-4L has the broadest tissue distribution, being detected in adult spleen, thymus, lymph node, peripheral blood leucocytes and bone marrow, and in fetal lung and liver. A soluble Fc chimaeric protein containing the hSiglec-5-4L extracellular domain binds in a sialic acid-dependent manner to glycophorin A on human erythrocytes and to alpha2-3- and alpha2-6-sialyllactose moieties. Domain deletion mutants of hSiglec-5(D1-4)-Fc reveal that the first three IgC2-SET domains are required for optimal binding, with adhesion being abolished if the first IgC2-SET domain is deleted. This indicates that each hSiglec-5 isoform will interact with sialic acid ligands and provides the first step towards defining structure-function relationships of hSiglec-5 isoforms.