Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA-damaging effects lead to apoptosis induction, but the early events of this signalling are still poorly understood. Our previous works have demonstrated the primordial role of NHE1 in B[a]P induced apoptosis in rat liver F258 epithelial cell line (Huc et al., Faseb J, 2004; Cancer Res, 2007). The aim of this study was to further elucidate how NHE1 is activated by B[a]P, and more specifically to investigate the role of plasma membrane (caveolae) in this activation. Two cholesterol-depleting agents, methyl-β–cyclodextrin and cholesterol oxidase, which are generally used for disrupting lipid rafts, were used in this study. Our results demonstrated that these two agents significantly decreased B[a]P (50nM)-induced apoptosis. Likewise, intracellular pH measurements showed that both the alkalinization and the acid efflux due to NHE1 activation upon B[a]P were also inhibited. This pointed to a possible role of lipid rafts in NHE1 activation. To gain further insight into such a role, NHE1 localization was studied after lipid raft extraction from cell lysates in Triton X-100 at 4°C on a sucrose density gradient. Our findings indicated that B[a]P not only affected raft composition, notably by decreasing cholesterol concentration, but also led to NHE1 delocalization outside these microdomains, these effects being prevented by cholesterol repletion. In total, our data provide evidence that B[a]P acts on microdomain composition, notably by altering cholesterol content, and that this alteration participates to NHE1 activation, thereby potentiating apoptosis.