Rapid Ca2+ efflux from intracellular stores during cardiac muscle excitation-contraction coupling is mediated by the ryanodine-sensitive calcium-release channel, a large homotetrameric complex present in the sarcoplasmic reticulum. We report here the identification, primary structure and topological analysis of the ryanodine receptor-calcium release channel from human cardiac muscle (hRyR-2). Consistent with sedimentation and immunoblotting studies on the hRyR-2 protein, sequence analysis of ten overlapping cDNA clones reveals an open reading frame of 14901 nucleotides encoding a protein of 4967 amino acid residues with a predicted molecular mass of 564 569 Da for hRyR-2. In-frame insertions corresponding to eight and ten amino acid residues were found in two of the ten cDNAs isolated, suggesting that novel, alternatively spliced transcripts of the hRyR-2 gene might exist. Six hydrophobic stretches, which are present within the hRyR-2 C-terminal 500 amino acids and are conserved in all RyR sequences, may be involved in forming the transmembrane domain that constitutes the Ca(2+)-conducting pathway, in agreement with competitive ELISA studies with a RyR-2-specific antibody. Sequence alignment of hRyR-2 with other RyR isoforms indicates a high level of overall identity within the RyR family, with the exception of two important regions that exhibit substantial variability. Phylogenetic analysis suggests that the RyR-2 isoform diverged from a single ancestral gene before the RyR-1 and RyR-3 isoforms to form a distinct branch of the RyR family tree.