Aims To investigate the cytotoxicity of beta-lapachone, a potent agent that may selectively target tumour cells, in retinoblastoma (RB) cell lines. Methods Growth inhibitory effects of betalapachone were evaluated in Y79, WERI-RB1, and RBM human retinoblastoma cell lines. Pro-apoptotic effects of beta-lapachone were evaluated in Y79 cells by detection of caspase 3/7 activity, by enzyme-linked immunosorbent assay for nucleosome fragments, and by cellular morphological analysis. Results Beta-lapachone induced significant dose-dependent growth inhibitory effects in all three retinoblastoma cell lines. The 50% growth inhibitory concentration (IC 50 ) of this agent was 1.9 lM in Y79 cells, 1.3 lM in WERI-RB1 cells, and 0.9 lM in RBM cells. Beta-lapachone also induced proapoptotic effects in RB cells. Treatment of Y79 cells with 1.9 lM beta-lapachone (IC 50 ) resulted in a peak, fourfold induction of caspase 3/7 activity at 72 h post-treatment; a peak, 5.6-fold increase in nucleosome fragments at 96 h posttreatment; and a peak, 1.7-fold increase in the frequency of apoptotic cells at 48 h posttreatment, relative to vehicle-treated controls. Conclusion Beta-lapachone induced potent cytotoxic effects in RB cell lines at low micromolar concentrations, suggesting this agent could be useful in the clinical management of RB.