Cellular distribution of the p27(KIP1) protein and its phosphorylation on threonine (T) 187 in mouse retinas from three stages of development, and retinoblastoma were examined. Retinas in C57Bl6 mice at embryonic day (E) 14, postnatal day (P) 1 and P11 were analyzed using immunohistochemistry with anti-p27(KIP1), threonine-187-phosphorylated p27(KIP1) (T187-phospho-p27), bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA) antibodies, and phosphorylated histon H3 (pHiston H3), which is a marker for cells in M phase. p27(KIP1) knockout (-/-) mice and human retinoblastoma were also analyzed. T187-phospho-p27 was detected in the outermost layer of the retina, whereas several neuroblastic cells expressed p27(KIP1) at E14. Many neuroblastic cells expressed BrdU in the middle layer. At P1, p27(KIP1) was detected in the ganglion cell layer and neuroblastic layer. T187-phospho-p27 was detected in the outermost layer, and that was localized in mitotic cells that also showed pHiston H3-positive. At P11, p27(KIP1) was detected in the inner nuclear layer, whereas T187-phospho-p27-positive or mitotic cells were not. BrdU positive nuclei were not detected in wild-type but were noted in the inner nuclear layer and the outer nuclear layer of the p27(KIP1) -/- mice retina at P11. In retinoblastoma, tumor cells formed numerous rosettes with Flexner-Wintersteiner rosettes. Several pHiston H3 -positive nuclei were noted in the tumor cells forming Flexner-Wintersteiner rosettes. Several T187-phospho-p27-positive nuclei were also detected in the mitotic cells forming Flexner-Wintersteiner rosettes. PCNA was expressed in rosette-forming cells. In conclusion, T187-phospho-p27(KIP1) was correlated with M phase of the cell cycle in the developing retina and retinoblastoma.