Earlier we showed using shock injury (OSI) that dysferlin modulates the coupling of excitation to Ca2+ release skeletal myofibers (Kerr et al., PNAS, 2013; Lukyanenko et al., Front. Physiol., 2022). Here we studied dysferlin-null A/J mouse FDB myofibers expressing fluorescent variants of nanodysferlins, created by the Sutton and Hirsch laboratories, that are missing different combinations of C2 domains, to assess their abilities to target t-tubules and to support normal Ca2+ signaling. cDNAs encoding nanodysferlins (reduced to 3 or 4 C2 domains plus the Fer/DysF domains) were provided by Drs. Hirsch and Sutton via the Jain Foundation. They were tagged at their N-terminus with Venus and electroporated into A/J FDB muscles. Expression of all four nanodysferlins was poor. When they were detected by confocal microscopy, they located in longitudinal membrane structures overlying the Z-disks typical of a compartment of the ER, not in the punctate structures typical of t-tubules. Nanodysferlins 364, 365, and 431 supported normal Ca2+ transient before OSI but behaved like untreated A/J fibers after OSI. Nanodysferlin 430 reduced the original amplitude of Ca2+ transients by ~20%, but supported their full recovery after OSI. All the nanodysferlins failed to suppress Ca2+ waves typical of CICR in myofibers exposed to OSI. Nanodysferlins with an N-terminal Venus tag express poorly in cultured A/J myofibers, mislocalize to the ER and fail to support normal Ca2+ signaling, especially after OSI. Supported by the Jain Foundation, and NIH (RO1 AR064268)