The evolution of multicellular animals (Metazoa) from their unicellular ancestors was a key transition that was accompanied by the emergence and diversification of gene families associated with multicellularity. To clarify the timing and order of specific events in this transition, we conducted expressed sequence tag surveys on 4 putative protistan relatives of Metazoa including the choanoflagellate Monosiga ovata, the ichthyosporeans Sphaeroforma arctica and Amoebidium parasiticum, and the amoeba Capsaspora owczarzaki, and 2 members of Amoebozoa, Acanthamoeba castellanii and Mastigamoeba balamuthi. We find that homologs of genes involved in metazoan multicellularity exist in several of these unicellular organisms, including 1 encoding a membrane-associated guanylate kinase with an inverted arrangement of protein-protein interaction domains (MAGI) in Capsaspora. In Metazoa, MAGI regulates tight junctions involved in cell-cell communication. By phylogenomic analyses of genes encoded in nuclear and mitochondrial genomes, we show that the choanoflagellates are the closest relatives of the Metazoa, followed by the Capsaspora and Ichthyosporea lineages, although the branching order between the latter 2 groups remains unclear. Understanding the function of "metazoan-specific" proteins we have identified in these protists will clarify the evolutionary steps that led to the emergence of the Metazoa.