This chapter describes two protocols for isolating total RNA from mycobacteria: one for extraction from in vitro cultures and one for extraction from in vivo. In these protocols, RNA is liberated from mycobacteria by disruption with small glass beads in the presence of Trizol to stabilize the RNA. The RNA is further purified with DNAse treatment and RNeasy columns. This protocol leads to microgram quantities of RNA from log-phase cultures.