Isoprene is a volatile metabolite of uncertain function in plants, animals, and bacteria. Here, we demonstrate that the isoprene-producing bacterium, Bacillus subtilis, contains an isoprene synthase activity that catalyzes dimethylallyl diphosphate-dependent isoprene formation. Although the enzyme was very labile, it was demonstrated in both permeabilized cells and in partially purified cell extracts. Its activity was optimal at pH 6.2, required low levels of a divalent cation, and appears distinct from chloroplast isoprene synthases. When grown in a bioreactor, B. subtilis cells released isoprene in three distinct phases; using permeabilized cells, it was shown that isoprene synthase activity rose and fell in parallel with each phase. These results suggest that isoprene synthesis is highly regulated in B. subtilis and further research in this model system may shed light on the role of isoprene formation in biological systems.