The Caribbean reef sponge Svenzea zeai was previously found to contain substantial quantities of unicellular photosynthetic and autotrophic microbes in its tissues, but the identities of these symbionts and their method of transfer from adult to progeny are largely unknown. In this study, both a 16S rRNA gene-based fingerprinting technique (denaturing gradient gel electrophoresis [DGGE]) and clone library analysis were applied to compare the bacterial communities associated with adults and embryos of S. zeai to test the hypothesis of vertical transfer across generations. In addition, the same techniques were applied to the bacterial community from the seawater adjacent to adult sponges to test the hypothesis that water column bacteria could be transferred horizontally as sponge symbionts. Results of both DGGE and clone library analysis support the vertical transfer hypothesis in that the bacterial communities associated with sponge adults and embryos were highly similar to each other but completely different from those in the surrounding seawater. Sequencing of prominent DGGE bands and of clones from the libraries revealed that the bacterial communities associated with the sponge, whether adult or embryo, consisted of a large proportion of bacteria in the phyla Chloroflexi and Acidobacteria, while most of the sequences recovered from the community in the adjacent water column belonged to the class Alphaproteobacteria. Altogether, 21 monophyletic sequence clusters, comprising sequences from both sponge adults and embryos but not from the seawater, were identified. More than half of the sponge-derived sequences fell into these clusters. Comparison of sequences recovered in this study with those deposited in GenBank revealed that more than 75% of S. zeai-derived sequences were closely related to sequences derived from other sponge species, but none of the sequences recovered from the seawater column overlapped with those from adults or embryos of S. zeai. In conclusion, there is strong evidence that a dominant proportion of sponge-specific bacteria present in the tissues of S. zeai are maintained through vertical transfer during embryogenesis rather than through acquisition from the environment (horizontal transfer).