Author Summary Anopheles gambiae, the principal Afrotropical mosquito vector for human malaria, uses olfaction to respond to chemical cues that are required for feeding, host preference, and mate selection. At the heart of this process is a large family of An. gambiae odorant receptors (AgORs) that respond to these olfactory cues in peripheral sensory neurons. We have now taken advantage of the relative simplicity of the larval olfactory system to develop a novel behavioral paradigm for individual larva that has been used together with gene silencing approaches in vivo to examine directly the role of AgORs in the olfactory system of An. gambiae. In addition to supporting and extending previous studies on the general olfactory response properties of AgORs, our data directly implicate the activity of specific AgORs in mediating the behavioral responses to the commercial insect repellent DEET and reveal the existence of at least two distinct olfactory signaling pathways in An. gambiae. One system depends directly on AgORs, while the other is AgOR-independent and requires the expression and activity of a newly identified family of candidate chemosensory genes, the An. gambiae variant ionotropic receptors (AgIRs). In addition to clarifying the mechanistic basis for olfaction in this system and the basis of DEET repulsion, these advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases.