Calreticulin is a Ca²⁺-binding chaperone localized mainly in the endoplasmic/sarcoplasmic reticulum in all higher organisms. To date, only one calreticulin isoform has been identified in human and mouse. Here we report a novel calreticulin isoform (Crt2) in human and mouse, with 53 (human) and 49% (mouse) identity to the previously identified calreticulin in respective species. The gene encoding the novel human calreticulin isoform spans 17 kb of genomic DNA and is expressed in testis, showing a similar expression as the chaperone calmegin. Phylogenetic analysis shows that two or more calreticulin (crt) genes are present both in plants and in mammals. The duplication of the crt gene in human and mouse suggests functional diversity, and variations in expression patterns among calreticulins. Two novel calreticulin (Crt2) isoforms, with high homology to the human and mouse calreticulin isoform (Crt2), were also identified in pig and rat via expressed sequence tags.