ßArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with ßarrestin1. Asp26 and Asp29 in the N-terminal domain of ßarrestin1 are critical for its binding to MEK1, whereas Arg47 and Arg49 in the N-terminal domain of MEK1 are critical for its binding to ßarrestin1. Wildtype FLAG-tagged ßarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A/D29A mutant does not. ERK-dependent phosphorylation at Ser412 was compromised in the D26A/D29A-ßarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated ßarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked ßarrestin1/ MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-ßarrestin1 in compromising the ERK-dependent phosphorylation of ßarrestin1. In addition, the MEK-disruptor peptide promoted the ability of ßarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the ß 2-adrenergic receptor. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.