BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous toxic environmental pollutants capable of inducing cell death. Intracellular pH plays a key role in the regulation of cell survival and death. Our previous works have demonstrated that intracellular alkalinization mediated by Na(+)/H(+) exchanger 1 (NHE-1) is a critical event involved in B[a]P-induced apoptosis. The aim of this study was to further elucidate the mechanisms of NHE-1 activation upon B[a]P exposure. METHODS: We tested the effects of plasma membrane cholesterol enrichment or depletion on B[a]P-induced NHE-1 activation related to apoptosis. We isolated cholesterol-rich plasma membrane microdomains to assess NHE-1 submembrane location and immunoprecipitated NHE-1 from the different sub-membrane fractions obtained to examine NHE-1 protein interactions during B[a]P-induced apoptosis. RESULTS: We found that NHE-1 is preferentially located in cholesterol-rich microdomains and that B[a]P activates NHE-1 via its relocation and binding of calmodulin outside these specialized plasma membrane microstructures; these events are necessary for the execution of the apoptosis-related intracellular alkalinization. CONCLUSION: Plasma membrane location of NHE-1 affects its protein interactions and apoptotic function.