Some organelles responsible for contraction consist of bundles of 2-4 nm filaments called nanofilaments. Such organelles are present in the longitudinal flagellum of Ceratium (Dinoflagellate): the R-fibre is the motor system for contraction and parallels the axoneme, which is responsible for wave generation. We used a highly sensitive polarization microscope developed by one of the authors to measure the birefringence of these nanofilament bundles during contraction in vivo. Our results show that the R-fibre gives a highly birefringent signal, retarding the polarization to much the same extent irrespective of the direction of polarization. By rotating the axis of the microscope compensator we confirmed that the birefringence is positive, suggesting that the bundles run parallel to the longitudinal axis of the flagellum. Conversely, when the compensator was rotated contrary to the direction of retardation, the bundle appeared dark (except when the organelle was in a fully contracted state). Experiments performed on detergent-treated and ATP-reactivated flagella show that a portion of the flagella regained activity with the addition of ATP in the presence of low Ca(2+) concentrations. This demonstrates the ability to reactivate flagellar motility after permeabilization and that axonemal microtubules were not responsible for the strong flagellar birefringence. Combined with complementary data from DIC microscopy of demembranated flagella and electron microscopy, these findings have led to the development of a model of the R-fibre and a comparison with other types of birefringent nanofilament bundles, such as the myoneme of Acantharia.