Fish myocytes continue to develop active tension when stretched to sarcomere lengths (SLs) on the descending limb of the mammalian length-tension relationship (Shiels et al. 2006). A greater length-dependent activation in fish than mammals could account for this, since the increase in Ca2+ sensitivity may overcome the tendency for force to fall due to reduced cross-bridge availability at SLs above optimal myofilament overlap. We stretched skinned fish and rat ventricular myocytes over a wide range of SLs, including those on the descending limb of the mammalian length-tension relationship. We found that fish myocytes developed greater active tension than rat myocytes at physiological Ca2+ concentrations at long SL. This was due to a higher Ca2+ sensitivity and a steeper relationship between Ca2+ sensitivity and SL in the fish myocytes. The greatest difference in Ca2+ sensitivity was found at the longest SL (2.7 µm), where pCa50 for the rat myocytes was 6.19 ± 0.07 pCa units compared with 6.54 ± 0.03 for fish myocytes (p < 0.05, One-way ANOVA with Student-Newman-Keul’s post-hoc, n = 6-8). We also investigated the diastolic properties of fish and rat myocytes at long SLs by measuring titin-based passive tension, titin isoform expression and titin phosphorylation. Fish myocytes produced higher titin-based passive tension despite expressing a higher proportion of a long N2BA-like isoform (38.0±2% of total vs 0% in rat, n = 4). However, titin phosphorylation in fish myocytes was lower than in rat, which may explain some of the difference in passive tension between species. The high level of titin-based passive tension and the differential phosphorylation of sarcomeric proteins in fish myocytes may contribute to the enhanced length-dependent activation and underlie the extended range of in vivo stroke volumes found in fish compared with mammals.