Regulation of reactive oxygen species and cytosolic free calcium ([Ca(2+)](cyt)) is central to plant function. Annexins are small proteins capable of Ca(2+)-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca(2+)](cyt) when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca(2+)-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca(2+) influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca(2+)-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd(3+) and had a Ca(2+)-to-K(+) permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca(2+) influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca(2+)-permeable transport pathways, regulate [Ca(2+)](cyt), and may function as peroxidases in vitro.