We have recently shown that elevated ERK activities stimulate proliferation of intestinal cells whereas low sustained levels of ERK activities correlate with G1 arrest and are required for expression of several enterocyte differentiation proteins. In an attempt to clarify how ERK1/2 regulates intestinal differentiation, the present study assessed the subcellular distribution and regulation of ERK proteins and activities in differentiated enterocytes. We report that (1) ERK1/2 and their upstream modulators Ras, p85 (PI-3K), Rac1, and MEK1 are found in the brush border; (2) brush border-associated ERK1/2 are stimulated by EGF and feeding; (3) immunoblotting of proteins phosphorylated on SP/K motif suggests the presence of ERK substrates in the brush border, one of which could be actin; and (4) pharmacological inhibition of ERK alters microvilli architecture. Our results suggest that ERK may play important roles in the control of microvilli structure and possibly, in brush border-associated responses in differentiated intestinal epithelial cells. (C) 2003 Elsevier Inc. All rights reserved.