Four different isotypes of beta-tubulin are known to be expressed in mammalian brain. Monoclonal antibodies against beta II, beta III, and beta IV were used to characterize the beta-tubulin isotypes in two ciliated bovine tissues: non-motile sensory cilia of retinal rod cells and motile cilia of tracheal epithelium. Retinal rod outer segment (ROS) connecting cilia and cytoskeletons were purified by density gradient centrifugation. This preparation contained more than 20 major protein components, as shown by dodecyl sulfate polyacrylamide gel electrophoresis. Electroblots were used to quantitate the relative amounts of beta II, beta III, and beta IV. The connecting cilium and cytoskeleton of the rod outer segment has less type III beta-tubulin than brain and more type IV. The ratio of beta IV to beta II in the ROS is nearly a factor of 8 larger than in brain. Electron microscopic immunocytochemistry showed extensive labeling of cilia by anti-type IV in thin sections of retinas and trachea, and also in purified ROS cilia and cytoskeletons. Labeling of cilia by anti-beta II was also observed, although in the purified ROS cilia and cytoskeleton, the anti-beta II labeling was primarily on amorphous non-ciliary material. The results suggest that both motile and non-motile cilia are enriched in the type IV beta-tubulin subunit.