Although wortmannin is extensively used in molecular signaling studies, its stability in tissue culture medium has not been assessed precisely. Therefore, we used high-performance liquid chromatography (HPLC) and mass spectrometry (MS) to characterize the decomposition of wortmannin in five commonly used media. Wortmannin was added to medium alone or to medium supplemented with 10% unheated or heat-inactivated fetal bovine serum and incubated at 37 degrees C. After 0, 5, 10, 20, 35, and 60 min, wortmannin remaining in the medium was quantified, and its decay constant and half-life were calculated. In all media, wortmannin decomposed monoexponentially, with half-lives between 8 and 13 min. HPLC/MS indicated that wortmannin decomposed to materials with m/z 447, 433, 373, and 313. Acidification of material produced by incubation of wortmannin in tissue culture medium or 1 microM NaOH converted the material with m/z 447 back to one that cochromatographed with and had an m/z (429) identical to that of wortmannin. Therefore wortmannin is much less stable in tissue culture medium than previously thought although some apparent loss of wortmannin reflects reversible, pH-dependent opening of the lactone ring of wortmannin. This rapid and complex decomposition of wortmannin argues for care being taken in how it is used in in vitro studies.