The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF1) ATPase activity was investigated through an examination of the effects of tentoxin [a toxin produced by Alternaria alternate] on electrophoretically-separated CF1 ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes" upon electrophoresis but none of the isozymes were tentoxin sensitive. CF1 isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. and Gray and the CF1-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF1 for tentoxin-induced chlorosis.