Agroinfiltration is a versatile, rapid and simple technique that is widely used for transient gene expression in plants. In this chapter we focus on its use in molecular plant pathology, and especially for the expression of plant resistance (R) and fungal avirulence (Avr) (effector) genes in leaves of Nicotiana benthamiana. Co-expression of an R gene with the corresponding Avr gene triggers host-defence responses that often culminate in a hypersensitive response (HR). This HR is visible as a necrotic sector in the infiltrated leaf area. Staining of the infiltrated leaves with trypan blue allows visual scoring of the HR. Furthermore, fusion of a fluorescent tag to the recombinant protein facilitates determination of its sub-cellular localization by confocal microscopy. The matching gene pair I-2 and Avr2, respectively from tomato and the fungal root-pathogen Fusarium oxysporum f. sp. lycopersici, is presented as a typical example.