Stage 16+ endomesodermal caudal explants from embryos of the zebrafish, Brachydanio rerio (Hamilton-Buchanan), were maintained in vitro for up to 48 h at 24 ± 2 °C. The development of Kupffer's vesicle by multivesicular coalescence and subsequent atrophy is described. Kupffer's vesicle in the absence of periblast and epidermal ectoderm is shown to be mesodermal in origin and not a remnant of the archenteron. The vesicle similarly would appear to play no role in the mediation of nutrients from the yolk via a modified periblast under conditions of culture.