Purpose. To identify and characterize retinoblastoma protein (pRb) binding proteins that may influence retinoblast proliferation and retinal pigment epithelial cell survival. Methods. The yeast two-hybrid system was used to screen a bovine retinal cDNA library and to characterize positive clones. DNA sequencing and site-directed mutagenesis were used for further analysis. Co-immunoprecipitation experiments were used to confirm the results of the two-hybrid system in vivo. Results. In the two-hybrid system, Protein Phosphatase 1α1 (PP1α1) binds the retinoblastoma protein. Unlike several other pRb binding proteins, PP1α1 binds only weakly to the Rb family member p107, and does not demonstrate detectable binding to p130. Confirming the two-hybrid results, endogenous PP1 in a human retinal pigment epithelial (RPE) cell line co-immunoprecipitates with endogenous pRb but not p107 or p130. Site directed mutagenesis of two pRb binding motifs in PP1α1 from LXSXE to LXCXE leads to slight increases in its two-hybrid interaction with pRb but does not alter its binding preference for pRb over the other family members. The complete sequence of bovine PP1α1 is reported. Conclusion. The strong two-hybrid interaction between PP1α1 and pRb, but not p107 or p130, suggests that the phosphorylation status of members of the pRb family may be regulated by different phosphatases, contributing to fine control of cell cycle progression. Conversely, PP1 activity may be specifically regulated by pRb and not p107 or p130. Mutagenesis studies suggest that PP1α1's LXSXE motif is not responsible for its binding preference for pRb over p107 and p130. Disruption of the PP1-pRb interaction may influence retinoblastoma tumorigenesis as well as RPE cell proliferation and survival.