The presence of either calreticulin (CR) or calsequestrin (CS-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca(2+)-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn(2+) (i.e., under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca(2+). Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca(2+)-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.