Synthesis of the aroma compounds of virgin olive oil (VOO) occurs through the lipoxygenase (LOX) pathway comprising mainly the actuation of LOX and hydroperoxide lyase (HPL) enzymes. The aim of this work was to determine whether the cleavage of polyunsaturated fatty acid hydroperoxides catalyzed by HPL is a limiting factor for the biosynthesis of VOO volatile compounds during the oil extraction process. For this purpose, HPL activity and the availability of substrates for this activity were modified during the oil extraction process from olive fruits of cultivars Arbequina and Picual, which give rise to oils with quantitatively different volatile profiles. Experimental data suggest that the HPL enzyme activity is just slightly limited during the oil extraction process in both cultivars, being this limitation apparently more significant during the processing of Arbequina fruits than of Picual fruits. However, this difference in HPL limitation seems to be more related to the differences in the amount of hydroperoxides produced in each cultivar than to the level of HPL activity during the olive processing.