With the broad objective of developing a heterologous expression system for the mammalian olfactory signaling pathway, we have engineered yeast cells in which the mammalian olfactory signaling pathway is genetically integrated. Our results demonstrate that the prototypic "olfactory yeast" strain WIF-1alpha can sense and report the presence of defined chemical agents through the engineered mammalian olfactory system. In this heterologous Saccharomyces cerevisiae-based expression system, the primary components of the mammalian olfactory signaling pathway have been engineered, and signaling by the rat olfactory receptor is coupled to the expression of green fluorescent protein. By shuttling a library of olfactory receptor ligand-binding pockets into the pre-engineered signaling units of WIF-1 yeast cells, we further demonstrate the ability of these olfactory yeast cells to detect 2,4-dinitrotoluene. Using this approach, our results have identified the novel rat olfactory receptor Olfr226 as a 2,4-dinitrotoluene-responsive receptor. Genetic integration of a highly discriminatory olfactory system into biologically stable and biochip-adaptable yeast cells, as presented here, can provide an ideal targeted chemosensing platform for detecting diverse chemical molecules. In addition to their potential use in deorphanizing the superfamily of olfactory receptors, the engineered olfactory yeast cells should be amenable for high-throughput screening to identify receptor-specific molecular targets.