The technique of in situ RNA hybridization provides a means of examining RNA expression within individual parasites at many stages of development in both the vertebrate and mosquito hosts. The protocols described in this chapter have been developed with the aim of preserving the morphology of the parasites within the infected host cells or tissues, so that many examples of each parasite stage can be identified and observed in a single experiment. The subcellular morphology of the parasite is also preserved, and the localization of abundant RNA transcripts can be directly visualized ( 1 , 2 ; Fig. 1 ). The parasite samples are extremely simple to prepare and do not require specialized purification procedures; in situ hybridization has, for example, been used successfully to demonstrate the expression patterns of a number of sexual-stage specific genes within gametocytes present in smears of a few microliters of infected blood ( 1 , 3 - 5 ). Fig. 1.? In situ detection of Pbs21 and ribosomal RNA in P. berghei parasites. Female gametocytes (fg), schizonts (s) and ookinetes (ook) present within smears of P. berghei-infected blood ( 1 ) or ookinete cultures ( 2 ) were cohybridized with biotin-labeled rRNA ( A ) and digoxigenin-labeled Pbs21 (B) antisense RNA probes. Signals were detected with rhodamineconjugated a ntidigoxigenin and fluorescein-conjugated antibiotin antibodies. Ribosomal RNA is detected throughout the cytoplasm of all parasite stages, but is not detected in nuclei (arrowed); the morphology of the parasites is, therefore, clearly visible. Pbs21 mRNA, by contrast, is detected in a stage-specific manner, only within female gametocytes and ookinetes, and is visible as punctate staining.