An alternative mevalonate-independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.