The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium. Keywords: 185/333 proteins; sea urchin; invertebrate immunity; coelomocytes; intestine; transmission electron microscopy The immune system of sea urchins lacks antibody-based adaptive immune responses. 1,2 However, there are strong similarities between other components of sea urchin and vertebrate immune systems. The sea urchin genome revealed broad homologies between the immune response genes of these deuterostome invertebrates and those of vertebrates. 3 Many of the sea urchin gene families that are putatively involved in pathogen recognition are highly diversified compared with their vertebrate homologues. 4 It has been suggested that gut associated pathogenesis and regulation of normal gut flora might have driven the expansion of these immune response gene families in sea urchins. 4 The 185/333 gene family is unique to the sea urchins and lacks homology to all other gene families identified thus far. Nair et al. 5 first revealed the importance of 185/333 molecules in sea urchin immune responses. They showed that 185/333 mRNA expression increased by up to 75-fold in immunologically activated coelomocytes, so that over 60% of sequences in a subtracted cDNA library of immune response expressed sequence tags were 185/333 mRNAs. These results suggest a fundamental role for 185/333 proteins in the immune responses of sea urchins. Further studies revealed high levels of variability within this new gene family 5,6 that are caused by the insertion or deletion of short sequence stretches called elements, single nucleotide substitutions and sequence repeats. Proteomic analyses indicate that each sea urchin is capable of expressing a diverse repertoire of 185/333 proteins and that the suite of 185/333 proteins expressed differs when sea urchins are chal...