Intraflagellar transport (IFT) refers to the bi-directional movement of particles and associated cargo along the axonemes of eukaryotic flagella and cilia. To provide a new perspective on the morphology of IFT particles, their association with the axoneme, and their composition, we have used immunogold localization coupled to detection via scanning electron microscopy. Here we co-localize in the Chlamydomonas flagellar axoneme polypeptides labeled with specific antibodies. Chlamydomonas EB1 localizes to the distal tip of the axoneme, as expected from previous immunofluorescent data (Pedersen et al. Curr Biol2003;13(22):1969-1974), thus demonstrating the utility of this approach. Using antibodies to IFT-related polypeptides, particles can be identified associated with the axoneme that fall into one of two classes: The first class is composed of IFT particles labeled with polyclonal antibodies to kinesin-2 and monoclonal antibodies to either IFT139 (an IFT complex A polypeptide) or IFT172 (a complex B polypeptide). The second class is comprised of particles that label with antibodies to IFT139 alone; thus, discrete particles are present associated with the axoneme that are composed only of complex A polypeptides. When IFT particles were purified by sucrose gradient ultracentrifugation, they appeared as more or less spherical aggregates of varying dimensions labeled with antibodies to IFT139 and to the motor protein kinesin-2. By contrast, isolated IFT particles that were labeled with IFT172 antibodies were not labeled with kinesin-2 antibodies. The data are discussed in terms of the total polypeptide composition of an IFT particle and the interaction of the particles with the motors that power IFT.