The subcellular distribution of plasma-membrane-type Ca2+-transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner-membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)-enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+-transporting ATPase (molecular mass approximately 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116-kDa Ca2+-transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue-specific manner: the PM pump was dominant in leaves, while the inner-membrane pump was expressed at a higher level in the roots. For the purification of calmodulin-binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116 kDa was partially sequenced.