The pbuE (ydhL) gene from Bacillus subtilis is known to encode the purine base efflux pump, and its expression is controlled by an adenine-dependent riboswitch. We cloned the pbuE gene from Bacillus amyloliquefaciens and examined gene expression by its own cis-acting regulatory elements in Escherichia coli. Regulation of pbuE expression, previously found in B. subtilis, was retained in this heterologous expression: it was induced by adenine and activated by a mutation in the 5' untranslated region, which disrupted transcription termination. This observation supports the model that the adenine-dependent riboswitch directly regulates pbuE expression, without requiring additional factors. Overexpression of the PbuE pump conferred upon the E. coli strain resistance to higher concentrations of inosine, adenosine and guanosine, and increased exogenous inosine accumulation by E. coli cells deficient in purine nucleoside phosphorylase. Overexpression of the PbuE pump also enhanced hypoxanthine excretion by the E. coli hypoxanthine-producing strain and inosine excretion both by the E. coli and B. amyloliquefaciens nucleoside-producing strains. Thus, for the first time, we obtained direct evidence for the involvement of PbuE in efflux of not only purine bases, but also purine ribonucleosides. A possible new role for the pump in cell physiology is discussed.