Four different isotypes of β‐tubulin are known to be expressed in mammalian brain. Monoclonal antibodies against β_II, β_III, and β_IV were used to characterize the β‐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 protein components, as shown by dodecyl sulfate polyacrylamide gel electrophoresis. Electroblots were used to quantitate the relative amounts of β_II, β_III, and β_IV. The connecting cilium and cytoskeleton of the rod outer segment has less type III β‐tubulin than brain and more type IV. The ratio of β_IV to β_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 cyoskeletons. Labeling of cilia by anti‐β_II was also observed, although in the purified ROS cilia and cytoskeleton, the anti‐β_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 β‐tubulin subunit. © 1993 Wiley‐Liss, Inc.