Methylbutenol (2-methyl-3-buten-2-ol) is an abundant volatile organic compound released from Western U.S. pines. To understand the mechanism of methylbutenol formation, we developed a sensitive gas chromatographic assay for its detection and determined that needles of gray pine (Pinus sabiniana) contain an enzyme that catalyzes the synthesis of methylbutenol from dimethylallyl diphosphate (DMAPP). The methylbutenol synthase activity was partially purified; its pH optimum was 7-8, and, like other prenyl diphosphate utilizing enzymes, it was dependent on the presence of a divalent cation, preferably Mn2+. The enzyme also required K+ or NH4+ for activity. The Km values for DMAPP and Mn2+ were about 4.8 and 6 mM, respectively. Geranyl diphosphate was not a substrate for the enzyme, so it is distinct from linalool synthase, a plant enzyme that catalyzes an analogous reaction. The methylbutenol synthase reaction may be responsible for the majority of light-dependent methylbutenol production by many pine species in the Western United States.