This review is devoted to the state-of-the-art strategies for the in vivo whole-transcriptome analysis of intracellular pathogens. The methods for the initial enrichment with bacterial RNA are discussed in detail, including the hybridization-based approaches, as well as the methods for cDNA synthesis taking into account the specific features of prokaryotic RNA and the methods for bacterial cDNA analysis, in particular, high-throughput RNA sequencing. The described methods are illustrated with an analysis of Mycobacterium tuberculosis transcriptome in different infection models (cell lines, infected animal tissues and organs, and human lung surgical samples). The advantages and limitations of the discussed methods are discussed. © 2010 Pleiades Publishing, Ltd.