Temperature-sensitive (ts) cell cycle mutant mouse cell, tsFT20, is deficient in DNA polymerase alpha activity to initiate DNA replication at replicon origins. Here, we analyzed phenotypes concerning growth control genes in the arrested tsFT20 cells. Analysis of cyclins showed that expression levels of cyclin D1, which is essential for G1/S transition, remarkably decreased in the mutant cells after temperature up-shift. Further we examined phosphorylation states of retinoblastoma protein (pRB) in the cells. Though the tsFT20 cells arrested in G1/S-S phase at nonpermissive temperature (Eki et al., (1990) J. Biol. Chem. 265 26-33), a large proportion of pRB was found as an underphosphorylated growth-suppressive form in the arrested cells. In revertant cell lines of tsFT20, pRB was not underphosphorylated even at nonpermissive temperature. The pRB underphosphorylation occurred later than the decrease of mRNA levels of cyclin D1, thus the underphosphorylation may be caused by the decrease in amount of cyclin D1 protein. These results indicated that the mutational inactivation of DNA polymerase alpha evokes phenotypes in which the inhibitory machinery of G1/S transition has been turned on.