Despite ongoing interest into the architecture, biochemistry, and physiology of the visual systems of the xiphosuran Limulus polyphemus, their ontogenetic aspects have received little attention. Thus, we explored the development of the lateral eyes and associated neuropils in late embryos and larvae of these animals. The first external evidence of the lateral eyes was the appearance of white pigment spots-guanophores associated with the rudimentary photoreceptors-on the dorsolateral side of the late embryos, suggesting that these embryos can perceive light. The first brown pigment emerges in the eyes during the last (third) embryonic molt to the trilobite stage. However, ommatidia develop from this field of pigment toward the end of the larval trilobite stage so that the young larvae at hatching do not have object recognition. Double staining with the proliferation marker bromodeoxyuridine (BrdU) and an antibody against L. polyphemus myosin III, which is concentrated in photoreceptors of this species, confirmed previous reports that, in the trilobite larvae, new cellular material is added to the eye field from an anteriorly located proliferation zone. Pulse-chase experiments indicated that these new cells differentiate into new ommatidia. Examining larval eyes labeled for opsin showed that the new ommatidia become organized into irregular rows that give the eye field a triangular appearance. Within the eye field, the ommatidia are arranged in an imperfect hexagonal array. Myosin III immunoreactivity in trilobite larvae also revealed the architecture of the central visual pathways associated with the median eye complex and the lateral eyes. Double labeling with myosin III and BrdU showed that neurogenesis persists in the larval brain and suggested that new neurons of both the lamina and the medulla originate from a single common proliferation zone. These data are compared with eye development in Drosophila melanogaster and are discussed with regard to new ideas on eye evolution in the Euarthropoda. Developmental Dynamics 235:2641-2655, 2006.