The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3a(KI) (containing amino acids C-19, K-80 and I-103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3a(EM) (S-19, E-80 and M-103). Only the AVR3a(KI) protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3a(KI) carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3a(KI) is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3a(KI) and AVR3a(EM) proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3a(EM) protein instability. AVR3a(KI) is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3a(KI), which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3a(KI) and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K-80 and I-103, of mature AVR3a contribute to the effector functions.