Previous results had suggested that Ca(H2PO4)2may be directly inhibitory to host penetration from appressoria ofErysiphe graminisf. sp.hordeiand that Ca and P are found in much higher amounts in oversized papillae (resistant to penetration) than in normal papillae (not resistant to penetration). In the present study, chlortetracycline was used as a calcium-selective fluorescent probe to examine the occurrence and distribution of calcium in papillae of barley coleoptile epidermal cells. When stained with chlortetracycline, normal and oversized papillae fluoresced bright yellow, indicating the presence of calcium. Oversized papillae, produced in the presence of calcium phosphate solution, could be either poststained or stained during papilla formation by including chlortetracycline in the incubation medium. Such attempts to stain normal papillae during their formation in the presence of calcium nitrate resulted in the formation of oversized papillae similar in appearance to those produced in the presence of Ca(H2PO4)2; however, penetration from appressoria was not inhibited by chlortetracycline. In some cases, this treatment resulted in the formation of chlortetracycline-fluorescent wall appositions on lower periclinal cell walls far removed from the fungus; these appositions were interpreted as calcium-containing wound plugs. None of the chlortetracycline-associated phenomena were seen without calcium in the incubation medium. These results confirm the occurrence and demonstrate the distribution of calcium in papillae and show that chlortetracycline, like phosphate, can act in conjunction with calcium to enhance secretion of papilla material.