Calsequestrin (CASQ) is an acidic, high capacity Ca2+-binding protein located within the terminal cisternae of the sarcoplasmic reticulum (SR). CASQ1 is the major SR Ca2+- buffer in fast muscle fibers, whereas CASQ2 predominates in cardiomyocytes and slow muscle fibers. Mice devoid of CASQ were generated by crossing mice lacking CASQ1 (Paolini et al J Physiol 2007, 583, 767) and mice lacking CASQ2 (Knollman et al J Clin Inv 2006, 116, 2510). When studied ex vivo, fast muscles (EDL) showed an earlier decline in tension during tetanic stimulation than WT fibers. This was not the case in slow muscle, soleus. Such decline of the developed tension was compatible with the decay of free cytosolic calcium during repeated stimulation observed using Fura-2 in single intact fast FDB muscle fibers. Single permeabilized fibers from EDL and tibialis anterior showed a reduction of the amount of calcium released by 30 mM caffeine, which is supposed to release about 80% of SR calcium (Murphy et al. J Physiol 2009, 587, 443), thus suggesting a depletion of SR. The depletion was confirmed by measurements of SR fee [Ca2+] using a FRET-based indicator (D1ER) genetically targeted to the SR. During contraction a massive/marked reduction in intraluminal free [Ca2+] was observed to a level close to full depletion.The results are consistent with the function of CASQ as a intraluminal SR buffer. CASQ1/2 null mice lack sufficient compensation for the loss in buffering power by other SR proteins. The diversity between slow and fast muscles might be attributed to the presence of parvalbumin as a relevant sink of calcium in cytosol of fast but not slow muscles (Celio et al Nature 1982, 297, 504).