Prenylated proteins undergo a series of post-translational modifications, including prenylation, proteolysis, and methylation. Collectively, these modifications generate a prenylcysteine methylester at the carboxyl terminus and modulate protein targeting and function. Prenylcysteine methylation is the only reversible step in this series of modifications. However, prenylcysteine alpha-carboxyl methylesterase (PCME) activity has not been described in plants. We have detected a specific PCME activity in Arabidopsis thaliana membranes that discriminates between biologically relevant and irrelevant prenylcysteine methylester substrates. Furthermore, we have identified an Arabidopsis gene (At5g15860) that encodes measurable PCME activity in recombinant yeast cells with greater specificity for biologically relevant prenylcysteine methylesters than the activity found in Arabidopsis membranes. These results suggest that specific and non-specific esterases catalyze the demethylation of prenylcysteine methylesters in Arabidopsis membranes. Our findings are discussed in the context of prenylcysteine methylation/demethylation as a potential regulatory mechanism for membrane association and function of prenylated proteins in Arabidopsis.