Curie-point pyrolysis/triple quadrupole mass spectrometry and micro-tube furnace pyrolysis/quadrupole ion trap mass spectrometry have been used to detect dipicolinic acid (DPA) in sporulated whole bacteria. DPA in whole cells of sporulated Bacillus anthracis reacted in situ during pyrolysis with tetramethylammonium hydroxide to form the dimethyl ester derivative of DPA, dimethyl-2,6-dipicolinate (mDPA). The mDPA was identified by its positive-ion electron ionization fragmentation pattern and confirmed with tandem mass spectrometry. In an oxidative pyrolysis/quadrupole ion trap instrument, the mDPA mass spectrum showed characteristic positive-ion electron ionization fragmentation along with a significant [M+1]+ ion due to self-chemical ionization. The characteristic collision-induced dissociation fragments of mDPA were used to establish the presence of sporulation in B. anthracis whole cells at a concentration of 2.2 x 10(7) CFU (colony-forming units)/mL using the triple quadrupole instrument. The total time for analysis, including sample preparation, was less than 10 minutes for both instruments.