The lower deuterostomes, including the echinoderms, possess an innate immune system that includes a subsystem with similarities to the vertebrate complement system. A homologue of the central component of this system, C3, has recently been identified in the purple sea urchin, Strongylocentrotus purpuratus, and is called SpC3. We determined previously that coelomocytes specifically express the SpC3 gene (Sp064); however, the sea urchin has at least four different types of coelomocytes: amoeboid phagocytes, red spherule cells, colorless spherule cells, and vibratile cells. To determine which of these subpopulations expresses Sp064 and produces SpC3, coelomocytes were separated by discontinuous gradient density centrifugation. Relatively homogenous fractions were obtained consisting of the four major cell types in addition to two types of amoeboid phagocytes with different densities and distinct morphologies. Analysis of proteins from separated cell subpopulations by Western blot and analysis of gene expression by RT-PCR revealed that phagocytes express the gene and contain the protein. Immunolocalization showed that SpC3+ phagocytes are present as subsets of both the low- and high-density subpopulations of phagocytes; however, the subcellular localization of SpC3 is different in these two subpopulations.