To address the mechanisms of ciliary radial spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The radial spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S radial spoke complexes competent to bind spokeless axonemes in vitro. Thus, pf27 is not defective in radial spoke assembly or docking to the axoneme. Rather, our results suggest that pf27 is defective in the transport of spoke complexes. During ciliary regeneration in pf27, radial spoke assembly occurs asynchronously from other axonemal components. In contrast, during ciliary regeneration in wild-type Chlamydomonas, radial spokes and other axonemal components assemble concurrently as the axoneme grows. Complementation in temporary dikaryons between wild-type and pf27 reveals rescue of radial spoke assembly that begins at the distal tip, allowing further assembly to proceed from tip to base of the axoneme. Notably, rescued assembly of radial spokes occurred independently of the established proximal radial spokes in pf27 axonemes in dikaryons. These results reveal that 20S radial spokes can assemble proximally in the pf27 cilium but as the cilium lengthens, spoke assembly requires transport. We postulate that PF27 encodes an adaptor or modifier protein required for radial spoke-IFT interaction. © 2013 Wiley Periodicals, Inc.