The neonatal isoform of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1b) is a Ca2+ pump with a well-known developmentally regulated transcript level but an undefined protein expression and function. Specific antibodies were generated to show that SERCA1b is exclusively expressed in myoblasts and myotubes of cultured and regenerating muscle. However, the SERCA1b protein was not detectable in normal adult fast and slow muscles. Studies of the in vitro differentiating myogenic cell lines C2C12 and sol8 showed that SERCA1b is the main SERCA1 protein isoform induced during differentiation and that it is found in the myotubes. Remarkably in BC3H1 cells, which show incomplete differentiation and are reluctant to form myotubes, express the SERCA1b mRNA but not the corresponding protein. SERCA1b protein was also absent from stretched or denervated adult soleus, in spite of the fact that its mRNA level was upregulated. SERCA1b accounts for nearly the total of SERCA1 expression in the diaphragm of newborn mice, which suggests that the insufficient function and development of the diaphragm in the SERCA1 null mutant mice may be due to the lack of SERCA1b. Our studies point to an important regulation of SERCA1b expression at the protein level and hints to a role in the growth of the developing muscle.