AbstractCilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N‐hydroxy succinimido‐biotin (NHS‐LC‐biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X‐100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X‐100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin‐labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non‐biotinylated protein at 51 kd. Other membrane+matrix proteins at 126 and 76 kd bound streptavidin even from noniabeled trachea, but remained soluble. Several biotin‐labeled proteins distinct from those in the membrane fraction remained with Triton X‐100‐extracted axonemes. Streptavidin‐colloidal‐gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat diffe
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