ABSTRACT Proteins occurring in minor amounts with purified sperm flagellar doublet microtubules were identified and studied by SDS-gel electrophoresis. Methods were developed to solubilize selectively these minor components; electron microscopy (EM) of the fractionated products revealed possible locations of these proteins in the tubule. Doublet microtubules were prepared from sea-urchin (Echinus esculentus and Strongylo-centrotus droebachiensis) and scallop (Pecten maximus) sperm by dialysing flagellar axonemes against 2 mM Tris-0·2 mM EDTA-0·5 rπM DTT. EM indicates that these doublet tubule preparations retain at least 70% of their radial spokes; cross-sections show a globule or fibre applied to the inside wall of the A-tubule, across from the inner B-tubule junction. On SDS-gels these preparations separate into at least 10 minor bands, accounting for 20–30% of the total protein; the remaining 75 ±4% migrates as tubulin. For E. esculentus the molecular weights and relative amounts of these components are: Component Ee 8 (150000 Daltons; 1 %), 11 (114 000; 2·5%), 15 (8 9 000; 2%), 16 (8 0 000; 2·5%), 17 (7 4 000; 2%), 18 (6 9 000; 2%), 19 (66 000; 2 %), 21 (48 000; 4·5 %), 22 (45 000; 3 %) and 23 (44 500; 3 %). Treatment of sea-urchin tubules with 0·1–0·5 % sarkosyl, 0·1–0·3 M KSCN or 0·3–0·6 M KI results in the selective solubilization of: first, component 8 and some B-subfibre tubulin; second, components 11 and 23 and the remaining B-subfibre tubulin; third, most of the A-subfibre tubulin and components 17, 18 and 19. Thermal fractionation extracts none of these components, suggesting they are principally associated with the A-tubule. Finally 25–35 % of the original protein is resistant to solubilization, and appears in the EM as ribbons of 3 protofilaments with 16-nm axial repeats. The resistant ribbons contain components 15, 16, 21 and 22 (plus component 20 in S. droebachiensis) in addition to 25 ±4% of the total tubulin. The data support the existence of two stable moieties in each doublet tubule: (1) a ribbon of 3 protofilaments and (2) either a second ribbon of 3 protofilaments or an equivalent amount of tubulin in some other form. EM images suggest that one ribbon forms the lateral side of the A-tubule (e.g. protofilaments A1,2,3 or A13,1,2 in the model) and that the globule applied to A13 may be a multisubunit complex of remaining minor components. Treatment of scallop tubules with 0·3 M KSCN preferentially extracts α-tubulin, yielding ribbons 1–4 protofilaments wide. The significance of this finding is discussed.