Geosmin is a terpenoid, an earthy-smelling substance associated with off-flavors in water and wine. The biosynthesis of geosmin is well characterized in bacteria, but little is known about its production in eukaryotes, especially in filamentous fungi. The origin of geosmin in grapevine is largely attributable to the presence of Penicillium expansum on grapes. Herein, we describe the characterization of "gpe1", a gene encoding a cytochrome P450 monooxygenase probably involved in the biosynthesis of geosmin in this species. A gpe1knockout mutant of P. expansum M2230 lost the capacity to produce geosmin, while the genetically complemented mutant restored it. The deduced gpe1 protein sequence shows identities with other cytochrome P450 monooxygenases involved in diterpene biosynthesis. These enzymes catalyze the addition of hydroxyl groups to the diterpene compounds. gpe1protein could work in the same way, with sesquiterpenes as substrates. This gene seems to be only present in geosmin-producing Penicillium species. To our knowledge, this is the first characterization of a fungal gene encoding an enzyme involved in geosmin biosynthesis.