A high-energy focused microwave system for killing experimental animals was used to rapidly inactivate enzymes and prevent postmortem breakdown of adenine nucleotides and adenosine, thereby enabling accurate measurements of AMP, ADP, ATP and adenosine in rat brain. For comparison, purine levels were measured in brains of rats killed by decapitation, decapitation into liquid nitrogen, or in situ freezing of the brain with liquid nitrogen. Of the three microwave irradiation power levels used, 10, 6.0 or 3.5 kW, rats killed by 10 kW had the highest ATP levels (28.8 nmol/mg protein) and cellular energy charge value (0.8), and the lowest levels of AMP (2.2 nmol/mg protein) and adenosine (19.7 pmol/mg protein). Of the 6 brain regions studied, adenosine levels (pmol/mg protein) ranged from 10 in cerebral cortex to 170 in cerebellum of rats killed using 10 kW microwave irradiation and, for comparison, ranged from 840 in cerebral cortex to 2498 in striatum of rats killed by decapitation. Focused microwave killing permits precise and accurate measurements of purines in discrete regions of rat brain.