The Xenopus LIM class homeobox gene Xlim-3 was identified initially as a fragment isolated by polymerase chain reaction cloning with an embryonic cDNA library as template (Taira et al., 1992, Genes Dev. 6, 356-366). cDNA clones representing most of the Xlim-3 mRNA were isolated from an adult brain library. The predicted Xlim-3 protein contains two copies of the LIM domain, a homeodomain, and a C-terminal region rich in proline, glycine, and serine. RNA blot hybridization showed that Xlim-3 mRNA is detected in dorsal regions at neural tube and tailbud stages and in adults predominantly in the pituitary gland and weakly in the eye and brain. Whole mount in situ hybridization revealed that Xlim-3 mRNA is first detectable at the neural plate stage in the stomodeal-hypophyseal (pituitary) anlage and in the neural plate where labeled cells were found adjacent to the forming floor plate. In situ hybridization analysis on serial sections at later stages showed that embryonic Xlim-3 expression persists in the pituitary and pineal, as well as in some cells of the retina, hindbrain, and spinal cord. In the retina, Xlim-3 mRNA was only detected in a distinct sublamina of the inner nuclear layer, but not in dividing cells of ciliary margin. This discrete manner of Xlim-3 expression, especially persistent expression in the pituitary (before morphogenesis of the gland to adult), supports a role in the specification and maintenance of differentiation of distinct neuronal and neuroendocrine tissues.