INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to fix, prepare, and hybridize antisense RNA probes in ctenophore embryos and cydippid larvae, as well as how to detect the probes using an alkaline phosphatase-conjugated antibody and colorimetric substrates. Using these techniques, it is possible to determine which cells or tissues express the gene of interest. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.