Calcineurin, the Ca2+/calmodulin-dependant serine/threonine phosphatase is the target for the immunosuppressant drugs FK506 and cyclosporine-A. These established calcineurin inhibitors each require an immunophilin protein cofactor. Gossypol, a polyphenol produced by the cotton plant, inhibits calcineurin (IC50=15 microM), in a noncompetitive, reversible manner, and is independent of any cofactor. We found that gossypol acts by at least two mechanisms to inhibit calcineurin phosphatase activity. A calmodulin-independent form of calcineurin was less sensitive to inhibition by gossypol than native calcineurin (IC50=41 and 18 microM, respectively) indicating that gossypol may interfere with calmodulin binding. A fluorescence polarization based assay demonstrated that 100 microM gossypol reduced the affinity of calmodulin for calcineurin (from K(d)=2.4 to 250 nM). Inhibition of calcineurin phosphatase activity by gossypol could not be overcome by adding excess calmodulin or by testing the inhibition toward a calmodulin-independent calcineurin indicating that gossypol acts at a site different from the calmodulin-binding site. Gossypol decreased the affinity of calcineurin for immunosuppressant/immunophilin complexes only in the presence of calmodulin, indicating that gossypol blocks the effects of calmodulin binding to calcineurin. In addition, gossypol had a stimulatory effect on native calcineurin in the absence of calmodulin, possibly indicating a calmodulin mimetic effect. Gossypol exists in two enantiomeric forms which are reported to have different potency for cell toxicity. (+) and (-) gossypol had equivalent potency for inhibition of native and calmodulin-independent calcineurin phosphatase activity, and for inhibition of calmodulin binding. The inhibition of calcineurin by gossypol via multiple binding sites without stereo-specificity indicates that gossypol is not a specific calcineurin inhibitor.