Wounded strawberry fruit produces a diverse group of volatile compounds including aldehydes, alcohols, and esters derived from the lipoxygenase (LOX) and hydroperoxide lyase (HPL) pathways. Because the wound volatiles may play an important role in plant-fungal interaction, the goal of this study was to develop a greater understanding about the biosynthesis of the major wound volatile, trans-2-hexenal (t-2-H), produced by strawberry fruit upon wounding. To that end, composition and quantity of total and free fatty acids of control and wounded strawberry fruit were analyzed. In addition, activities of the key enzymes, LOX and HPL, and production of C6 aldehydes were determined. Intact strawberry fruit did not produce detectable t-2-H which is derived from alpha-linolenic acid (18:3). However, in response to wounding by bruising, strawberry fruit emitted t-2-H and its precursor cis-3-hexenal (c-3-H). The level of total lipid 18:3 in the fruit increased 2-fold in response to wounding, whereas free 18:3 declined slightly ( approximately 30%). At 10 min following wounding, fruit exhibited a 25% increase in LOX activity, which leads to the production of 13-hydroperoxyoctadecatrienoic acid (13-HPOT) from 18:3. The activity of HPL, which catalyzes formation of cis-3-hexenal from 13-HPOT, increased 2-fold by 10 min after wounding. Thus, during a 15 min period after wounding, free 18:3 substrate availability and the activity of two key enzymes, LOX and HPL, changed in a manner consistent with increased c-3-H and t-2-H biosynthesis.