The sarcomere, the smallest functional unit of the muscle fibre, is the site at which contraction occurs. During development sarcomere structure is established in a series of steps, starting with the initiation of actin polymerisation at protocostameres (1). The later stages, which include determination of sarcomere length, are regulated by muscle contraction. Studies have revealed that inhibition of contraction causes myofibril and sarcomere disassembly, whereas stimulation accelerates reassembly. Nonetheless the process of contraction-driven sarcomere assembly in vivo is not well understood. The role of contraction on thin filament (actin) length in the developing sarcomere was examined during embryogenesis using the zebrafish (Danio rerio) as a model. Contraction was inhibited in developing zebrafish embryos by treatment with the reversible anaesthetic (Tricaine, MS222) starting at 17 hpf. This time coincides with the development of functional neuromuscular junctions and the initiation of body movements. At 24 hpf, embryos were transferred either to medium without anaesthetic which restored movement (recovered) or maintained in anaesthetic to block movement (treated). Actin thin filaments were observed within the skeletal muscle of fixed, whole mount embryos using a fluorescently labelled phalloidin (Alexa Fluor® 488 Phalloidin) and confocal scanning laser microscopy (Leica SP5). Thin filament length was determined by measurement from the periphery of one H-zone to the adjacent H-zone. Treated, immobilised embryos showed a significant increase in actin/thin filament length (1.72±0.01µm) compared to control embryos (1.66±0.01µm) at 24 hpf (P=0.016, unpaired t-test). However, by 42 hpf there was no significant differences between the length of actin filaments in control, treated or recovered embryos (ANOVA). Our results show that the absence of contraction between 17 and 24 hpf results in lengthening of the thin filaments in sarcomeres in vivo. This time period coincides with spontaneous, alternating contractions of the trunk movements within the embryo. The purpose of the body movements is unknown but we propose that they could be important for the final stages of skeletal muscle development, i.e. sarcomere organisation. By 42hpf, thin filament length had been restored in both treated and recovered embryos. This suggests that whilst contraction plays a role, there are other mechanisms that may act to regulate actin filament length in vivo. Our study has contributed to our understanding of the pathways that regulate contraction driven myofibril alignment and sarcomere arrangement in vivo. This work may have direct application to clinical studies in which normal muscle function is perturbed (e.g. muscle wasting as observed during bed rest in human patients).