exoBrevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive Mountain Pine Beetle (Dendroctonus ponderosae). It is also found in other insects and the African elephant. Despite its significance, very little is known about its biosynthesis. A recent microarray analysis implicated a small cluster of three D. ponderosae genes in exo-brevicomin biosynthesis, two of which had identifiable open reading frames (Aw et al., 2010; BMC Genomics 11:215). Here we report further expression profiling of two genes in that cluster and functional analysis of their recombinantly-produced enzymes. One encodes a short-chain dehydrogenase that used NAD(P)(+) as a co-factor to catalyze the oxidation of (Z)-6-nonen-2-ol to (Z)-6-nonen-2-one. We therefore named the enzyme (Z)-6-nonen-2-ol dehydrogenase (ZnoDH). The other encodes the cytochrome P450, CYP6CR1, which epoxidized (Z)-6-nonen-2-one to 6,7-epoxynonan-2-one with very high specificity and substrate selectivity. Both the substrates and products of the two enzymes are intermediates in the exo-brevicomin biosynthetic pathway. Thus, ZnoDH and CYP6CR1 are enzymes that apparently catalyze the antepenultimate and penultimate steps in the exo-brevicomin biosynthetic pathway, respectively.