The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) uniquely contains three cGMP binding sites, but also has a 'degenerate' fourth site. The role of each cGMP-binding site in PfPKG activation remains unknown. We have analysed the effect of mutation of each cGMP-binding site (individually and in combination) on PfPKG activation in vitro. The most striking result was that mutation of cGMP site 3 resulted in a 10-49-fold increase in the K (a((cGMP))) value and a 45-55% decrease in maximal activity compared with wild-type. Mutations involving only cGMP-binding sites 1 and 2 had less effect on both the K (a((cGMP))) values and the maximal activities. These results suggest that, although all three cGMP-binding sites are involved in PfPKG activation, cGMP-binding site 3 has the greatest influence on activation. A mutation in the fourth, degenerate cGMP-binding site decreased PfPKG maximal activity by 40%, but did not change the K (a((cGMP))) value for the PfPKG mutant, suggesting that this site does not bind cGMP, but is required for full activation of PfPKG. The distinct activation properties of PfPKG from mammalian isoforms may be exploitable in the design of a parasite-specific inhibitor and development of a novel anti-malarial drug.