The assembly of the intestinal microvillus cytoskeleton was examined during the differentiation of enterocytes along the crypt-villus axis in adult chicken duodenum using light and electron microscopic immunolocalization techniques. Using antibodies reactive with villin, fimbrin, and the heavy chain (hc) of brush border (BB) myosin I (110K-calmodulin complex) and rhodamine-conjugated phalloidin as a probe for F-actin, we determined that while actin, villin, and fimbrin were all localized apically along the entire axis, BB myosin I (hc) did not assume this localization until the crypt-villus transition zone. In addition to their localization at the BB surface, all four proteins were present at significant levels along the lateral margins of enterocytes along the entire crypt-villus axis, suggesting that these proteins may be involved in the organization and function of the basolateral membrane cytoskeleton as well. The pattern of expression of the microvillar core proteins along the crypt-villus axis in the adult was comparable to that seen in the intestine of the late stage chicken embryo and suggests that a common program for brush border assembly may be used in both modes of enterocyte differentiation.