Many marine sponge species contain bacterial symbionts within the intercellular matrix. However, direct evidence on when in the course of evolution these symbioses might have begun is not available. Sponge-specific bacterial strains (296) were isolated from 36 sponge species from the three major sponge classes collected from seven widely separate geographical regions. Strains resembling these sponge-specific ones were not found among 202 ambient water strains. A considerable proportion (40%) of the sponge-specific strains showed common antigens in agglutination reactions against immune sera prepared from seven of them, whereas none of the non-specific bacterial strains (94) and none of the seawater bacteria (34) agglutinated in these sera. Furthermore, the sponge-specific bacteria did not agglutinate with five antisera prepared from control, non-specific, bacterial strains. The establishment of symbioses is a rare and conservative event and immunological cross-reactions are usually considered to be highly specific. This evidence, supported by the knowledge of sponge evolution and of the sparse bacterial populations in hexactinellid sponges, suggests that the symbioses may have commenced during the Precambrian, before the evolution of the extant sponge orders and classes.