Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon emitted from many plant species to the atmosphere, where it plays an important role in atmospheric chemistry. An enzyme extracted from aspen (Populus tremuloides) leaves was previously found to catalyze the Mg(2+)-dependent elimination of pyrophosphate from dimethylallyl diphosphate (DMAPP) to form isoprene (Silver, G. M., and Fall, R. (1991) Plant Physiol. 97, 1588-1591). This enzyme, isoprene synthase, has now been purified 4000-fold to near homogeneity. The enzyme had a native molecular mass of 98-137 kDa and isoelectric point of 4.7 and contained 58- and 62-kDa subunits, implying that it is a heterodimer. Partial amino acid sequences of the two subunits indicated they are closely related to each other and that they do not share a strong homology with any other reported proteins. The isoprene synthase reaction was dependent on Mg2+ or Mn2+, and the reaction products were shown to be isoprene and pyrophosphate with a stoichiometry close to 1:1. The Km for DMAPP was high at 8 mM, and the kcat of 1.7 s-1 was low, but similar to those of other allylic diphosphate-utilizing enzymes. It is argued that the isoprene synthase reaction may be much more efficient in vivo, where it is under light-dependent control. It seems probable that this unique enzyme, rather than non-enzymatic reactions, can account for the emission of hundreds of millions of metric tons of isoprene from plants to the global atmosphere each year.