1. We have used fluorescence confocal laser scanning microscopy to attain the three-dimensional (3-D) microstructure of perimysial collagen fibres over the range of sarcomere lengths (1.9-2.3 micrometers) in which passive force of cardiac muscle increases steeply. 2. A uniaxial muscle preparation (right ventricular trabecula of rat) was used so that the 3-D collagen configuration could be readily related to sarcomere length. Transmission electron microscopy showed that these preparations were structurally homologous to ventricular wall muscle. 3. Trabeculae were mounted on the stage of an inverted microscope and fixed at various sarcomere lengths. After a trabecula was stained with the fluorophore Sirius Red F3BA and embedded in resin, sequential optical sectioning enabled 3-D reconstruction of its perimysial collagen fibres. The area fraction of these fibres, determined from the cross-sections of seven trabeculae, was 10.5 +/- 3.9 % (means +/- s.d.). 4. The reconstructed 3-D images show that perimysial collagen fibres are wavy (as distinct from coiled) cords which straighten considerably as the sarcomere length is increased from 1.85 +/- 0.06 micrometer (near-resting length) to 2.3 +/- 0.04 micrometer (means +/- s.d., n = 4). These observations are consistent with the notion that the straightening of these fibres is responsible for limiting extension of the cardiac sarcomere to a length of approximately 2.3 micrometers.