Abstract: LIM homeodomain proteins Islet-1 and Lim-3 expression and their role in nervous tissue and endocrine glands have been reported; however, nothing is known concerning Islet-1 and Lim-3 expression in the developing pineal gland of the chick embryo. The aim of the present study was to determine the ontogeny of Islet-1 and Lim-3 expression in the developing pineal gland of chick embryo using immunohistochemistry. The results showed that Islet-1 and Lim-3 immunopositive cells were first detected in the pineal evagination of chick embryos at day 4 (E4) and E4.5 of incubation, respectively. In the later developing stages, both Islet-1 and Lim-3 immunopositive cells were consistently detected in the follicular and parafollicular pinealocytes throughout the pineal gland. The relative percentage of Islet-1 immunopositive (Islet-1+) cells relative to the total cells was about 6% at E4.5, and then kept increasing (P < 0.05) and reached about 40% by E12.5; this was followed by no obvious changes until the chicks were newly hatched. The change in Lim-3 immunopositive (Lim-3+) cell number was parallel to that of Islet-1, although Lim-3+ cell were significantly fewer than Islet-1+ cell numbers from E4.5 to E8.5 (P < 0.05). Dual immunohistochemical staining results showed that almost all the Lim-3+cells expressed Islet-1 at every stage examined, and about 90% of Islet-1+cells were proliferating cell nuclear antigen negative. These results suggest that both Islet-1 and Lim-3 may be involved in regulating the development and functional maturation of the pineal gland, although further studies are required in elucidating the functional roles of Islet-1 and Lim-3 and the related mechanisms.