MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. In the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that At1g80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of At1g80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of At1g80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.