The retinoblastoma protein (pRb) is a key regulator of cell growth, differentiation and survival. pRb(-/-) mice show abnormal neuronal cell death in the developing brain. The function of pRb is regulated by its phosphorylation state. In this study, the phosphorylation of pRb during retinoic acid (RA)-induced neuronal differentiation of P19 cells was examined using site-specific antibodies against pRb phosphorylated at Ser601, Ser605 and Ser773. Although pRb was hyperphosphorylated in undifferentiated P19 cells, Ser601 and Ser773 were not phosphorylated. Upon exposure to RA, however, these two sites became strongly phosphorylated. Cdk4 kinase activity was almost undetectable in undifferentiated P19 cells, but was strongly activated on exposure to RA. In contrast, Cdk2 kinase activity and the phosphorylation of Ser605 were observed in undifferentiated cells as well as in RA-treated cells. These observations suggest that Cdk2 and Cdk4 may phosphorylate different sites of pRb in vivo and that the two sites of pRb examined here are newly phosphorylated during RA-induced neuronal differentiation in P19 cells.