Introduction: Ca2+ release from mammalian sarcoplasmic reticulum (SR) via type 2 ryanodine receptor (RyR2) channels is a pivotal step in cardiomyocyte contraction. This Ca2+ release is regulated by several factors, including interaction of RyR2 channels with accessory proteins. The SR intraluminal proteins calsequestrin-2 (CASQ2) and histidine-rich Ca2+ binding protein (HRC) exert opposing effects on RyR2 gating: CASQ2 inhibits diastolic Ca2+ release, whereas HRC promotes RyR2 gating. HRC could act via interactions with the luminal domains of SR proteins, including RyR2, the SERCA2a Ca2+ pump and triadin (Zhang, Waddell & Jones (2015)). Despite their importance in cardiac health and disease, little is known about the evolution or roles of HRC proteins. The current study aims to address these deficits. Methods: HRC homologs were identified by BLASTP searches of protein databases from a wide range of organisms. The evolutionary relationships between these proteins were reconstructed using the Maximum Likelihood method. Conserved patterns shared between homologs were derived using PRATT software. Apparent mutation rates of HRC and other SR proteins were estimated from alignments and from Ka/Ks analyses. Candidate Ca2+-binding proteins (CBPs), staining purple with the cationic dye Stains-All, were partially isolated from chicken heart microsomes by stepwise potassium chloride washing, sodium carbonate (pH 11.2) extraction and heat denaturation (as mammalian HRCs are heat stable). Two chicken heart SR proteins, of 122 kDa and 325 kDa apparent molecular weight, were analysed by the Protein Identification Service (University of York, UK). Results and Discussion: HRC homologs were encoded within many genomes, including those of most vertebrates, sponges, molluscs, bacteria and archaea. These homologs clustered poorly in phylogenic reconstructions, even between closely related species, suggesting rapid rates of evolution. Analyses of apparent mutation rates support this hypothesis. A conserved pattern of cysteine residues in HRC homologs suggests a relationship with the ferredoxin superfamily and participation in redox events. Birds apparently lack HRCs: homologs were not detected in proteomes of 26 avian species, but all of these encoded homologs of triadin. Several candidate CBPs were detected in microsomal preparations from chicken heart. Of these, mass spectrometry data suggest that a 122 kDa protein is sarcalumenin, an SR CBP. The identity of other bird heart SR intraluminal CBPs, including counterparts of mammalian HRCs, are currently being investigated.