The biological and forensic use of ion-interaction reversed-phase high-performance liquid chromatography for the determination of hydrogen sulfide-derived methylene blue is evaluated by measuring the sulfide content in tissues from sulfide-treated mice. Various preparative conditions were examined. The determinations of background levels of sulfide from brain, liver and kidney were compared to sulfide levels from mice exposed to 60 micrograms g-1 sodium hydrosulfide. At the time of death, significant increases above background sulfide levels were measured for all three biological tissues. To evaluate its forensic potential, we used this sulfide detection methodology to evaluate comparatively the sulfide levels from fresh and frozen samples of brain, liver and kidney. The stability of sulfide levels obtained from frozen brain makes this tissue the most reliable tissue for forensic evaluation. Samples of brain, liver and kidney obtained within 24 h of death by sulfide intoxication had demonstrable elevations in sulfide concentration.