Long-term survival of microorganisms has been demonstrated by prokaryotes cultured from ancient halite, but previous results are controversial. Three genera of non-spore-forming halophilic Archaea were cultured from 22–34 k.y. old subsurface halite from Death Valley, California. Primary, brine-filled inclusions in this halite contained prokaryotic organisms in miniaturized starvation-survival forms and dead cells of the algal genus Dunaliella. The energy needed for protracted survival of halophilic Archaea, including repair of damaged DNA, may have been provided by glycerol and other carbon molecules leaked from Dunaliella cells. These results provide further evidence that fluid inclusions in halite are a favorable refuge for long-term survival of microorganisms, and indicate that the original depositional environment influences the distribution and viability of prokaryotes.