The alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) is a promising new drug for treating various types of cancer. Its mechanism of action is not yet fully understood but is related to the Rb tumor suppressor protein. In the present study, we investigated the role of decreased Rb expression levels for the antileukemic efficacy of ErPC3 in BV-173 and K-562 CML-derived cell lines. We used antisense technique to knock down Rb levels in the two cell lines in addition to ErPC3 treatment. Cells with reduced Rb expression showed a diminished sensitivity to ErPC3 exposure, as determined by MTT (BV-173 and K-562) and clonogenicity assays (K-562 only), if concentrations below the IC50 were used. The feasibility of Rb knockdown varied between BV-173 and K-562 cells, with the former being distinctly more sensitive than the latter. We conclude that sufficient Rb levels are important for the cytotoxic and anticlonogenic effects of ErPC3 at levels below the IC(50), but that higher concentrations of ErPC3 are less dependent on Rb status.