The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca(2+) from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA3) and 5 mM CaCl2 results in a 70-80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca(2+) withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca(2+). The addition of Ca(2+) stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [(35)S]methionine show that Ca(2+) withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca(2+), α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca(2+) influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca(2+) concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca(2+) while Ba(2+) is only 10% as effective. We conclude that Ca(2+) regulates the secretion of enzymes and other proteins from the aleurone layer of barley.