Commissureless (Comm) controls axon guidance across the Drosophila melanogaster midline by regulating surface levels of Robo, the receptor for the midline repellent Slit. Two different models have been proposed for how Comm regulates Robo: a 'sorting' model and a 'clearance' model, both based on studies using heterologous cells in vitro. Here, we test these two models in vivo. We establish a genetic rescue assay for Comm, and use this assay to show that midline crossing does not require the presence of Comm in midline cells, as proposed by the clearance model. Moreover, by monitoring the trafficking of a Robo-green fluorescent protein (GFP) fusion in living embryos, we demonstrate that Comm prevents the delivery of Robo-GFP to the growth cone, as predicted by the sorting model. It has also been suggested that Comm must be ubiquitinated by the Nedd4 ubiquitin ligase. We show here, however, that ubiquitination of Comm is not required for its function in vitro or in vivo, and that Nedd4 is unlikely to function in axon guidance at the midline.