Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon of uncertain function in Bacillus subtilis, and we hypothesized that it is an overflow metabolite produced during excess carbon utilization. Here we tested this idea for phase 2 of isoprene release, a phase that occurs during extracellular acetoin accumulation and its reassimilation. Phase 2 isoprene formation could be disrupted in three different ways, all related to acetoin metabolism. Disruption of a gene essential for acetoin biosynthesis (acetolactic acid synthase, alsS) blocked acetoin formation and led to cessation of phase 2 isoprene formation as well as a variety of pleiotropic effects related to loss of pH control. Growth of the alsS mutant with external pH control reversed most of these effects. Disruption of acetoin catabolism (acetoin dehydrogenase, acoA), also eliminated phase 2 isoprene formation and caused cells to transition directly from phase 1 to phase 3; the latter is attributed to amino acid catabolism. A third alteration of acetoin metabolism was detected in the widely used strain 168 (trpC2) but not in strain MS175, a trpC mutant constructed in the Marburg strain genetic background. Strain 168 exhibited slow acetoin assimilation compared to that of MS175 or the parental strain, with little or no isoprene formation during this growth phase. These findings support the idea that isoprene release occurs primarily when the rate of carbon catabolism exceeds anabolism and that this volatile hydrocarbon is a product of overflow metabolism when precursors are not required for higher isoprenoid biosynthesis. It is suggested that isoprene release might serve as a useful barometer of the rise and fall of central carbon fluxes during the growth of Bacillus strains in industrial bioreactors.