Background: The mechanisms of bilirubin neurotoxicity are poorly understood. We hypothesize that free bilirubin at concentrations found in preterm infants increases neuronal apoptosis. To test this hypothesis, we measured the expression of pro-apoptotic proteins in primary cultures of cerebellar granule neurons exposed to bilirubin using quantitative immunoblotting techniques. Methods: Cerebellar granule neurons were plated on poly-L-Lysine overnight, then treated with human serum albumin combined with bilirubin to produce physiologic concentrations of free bilirubin. Staurosporine was added to some cultures as a positive control. Cell lysates were prepared, and cleaved caspase 3 expression was determined using quantitative immunoblotting techniques. Results: Addition of physiologic concentrations of free bilirubin to cerebellar granule neurons increased the expression of cleaved caspase 3 in a dose dependent manner at 24 hours. Conclusions: Physiologic concentrations of free bilirubin induces apoptosis in cultured rat cerebellar granule neurons.