The stiffness (flexural rigidity) of live sperm flagella, Triton-demembranated flagella (axonemes), trypsin-digested axonemes, and doublet microtubules of the axonemes in echinoderms was determined from the relationship between their deformation when a stream of medium was applied and the viscous resistance of the medium acting on the flagellum. The stiffness of the flagellum beating in seawater was 5.8 x 10(-21) Nm2 for bending in the direction perpendicular to the beating plane and 4.2 x 10(-22) Nm2 for bending within the beating plane. A similar difference in stiffness from the difference in bending directions was found in reactivated flagella with 1 mM ATP. The stiffness of live flagella immobilized in CO2-saturated seawater and axonemes in ATP-free medium was similar to that of beating flagella for bending in the direction perpendicular to the beating plane. The stiffness of motionless flagella significantly decreased with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and vanadate. The trypsin-digestion of motionless axonemes did not change their stiffness. The stiffness of doublet microtubules was 1.4 x 10(-23) Nm2 in 0.1 mM ATP medium and 6.1 x 10(-23) Nm2 in ATP-free medium. These results suggest that doublet pairs lying parallel to the beating plane of the flagellum retain fewer cross-bridges than doublet pairs lying perpendicular to the beating plane.