The further biochemical characterization and subcellular localization of cardiotin, a high molecular weight (300 kDa) constituent of cardiac muscle, is described. Immunofluorescence assays revealed a colocalization of cardiotin and the Ca2+ pump SERCA2a in the longitudinal sarcoplasmic reticulum (SR). However, in contrast to SERCA2a, cardiotin is not detected in the junctional SR. Differential centrifugation experiments show that cardiotin cosediments with the microsomal fraction of swine heart, while differential extraction demonstrates that cardiotin is associated with the SR membranes. In the SR enriched cell fraction a 60 and a 100 kDa protein band are detected. Microsequence analyses of these two fragments showed a common amino-terminus of 14 amino acids, with great homology to amino acid positions 11-24 of human skeletal muscle alpha-actinin. Second generation antibodies directed to these specific fragments show the typical cardiotin pattern in cardiomyocytes and cross-reactivity amongst the respective antigens. Cardiotin did not colocalize with alpha-actinin, and alpha-actinin could not be detected in the microsomal SR fraction. Cardiotin therefore represents a new SR associated constituent.