Approximately half of all living oysters brood offspring in the inhalant chamber of their mantle cavities; the remainder are broadcast spawners which do not engage in parental care of young. Ostreid ovulation involves a complex behavioral sequence that results in the countercurrent passage of newly spawned eggs through the gills (ctenidia) and into the inhalant chamber. We constructed molecular and combinedevidence phylogenetic trees to test hypotheses concerning the directionality of parental care evolution, and the evolutionary significance of the trans-ctenidial ovulation pathway, in the Ostreidae. Representatives of all three ostreid subfamilies, together with gryphaeid and nonostreoidean pterioid outgroups, were sequenced for a 941-nucleotide fragment of the 28S ribosomal gene. Our phylogenetic analyses indicate that (1) the Ostreidae are robustly monophyletic, (2) broadcast spawning and larval planktotrophy are ancestral ostreid traits, (3) trans-ctenidial ovulation predates the evolution of parental care in ostreid lineages, and (4) brooding originated once in the common ancestor of the Ostreinae/Lophinae, involved a modification of the final behavioral step in the ancestral ovulation pathway, and has been retained in all descendent lineages. Our data permit an independent test of fossil-based ostreid phylogenetic hypotheses and provide novel insights into oyster evolution and systematics. 2000 Academic Press