The development of olfactory bulb projections that form the lateral olfactory tract (LOT) is still poorly understood. It is known that the septum secretes Slit1 and Slit2 which repel olfactory axonsin vitroand that inSlit1−/−;Slit2−/−mutant mice, the LOT is profoundly disrupted. However, the involvement of Slit receptors, the roundabout (Robo) proteins, in guiding LOT axons has not been demonstrated. We show here that both Robo1 and Robo2 receptors are expressed on early developing LOT axons, but that only Robo2 is present at later developmental stages. Olfactory bulb axons fromRobo1−/−;Robo2−/−double-mutant mice are not repelled by Slitin vitro. The LOT develops normally inRobo1−/−mice, but is completely disorganized inRobo2−/−andRobo1−/−;Robo2−/−double-mutant embryos, with many LOT axons spreading along the ventral surface of the telencephalon. Finally, the position of lot1-expressing cells, which have been proposed to be the LOT guidepost cells, appears unaffected inSlit1−/−;Slit2−/−mice and inRobo1−/−;Robo2−/−mice. Together, our results indicate that Robo1 and Robo2 directly mediate the repulsive activity of Slit receptors on LOT axons, and are required for normal guidance of these axonsin vivo.