The plasma membrane of the kidney brush border is composed of two compositionally distinct microdomains, microvilli and clathrin-coated pits. To study their assembly we have immunolocalized brush border marker proteins in the developing proximal tubule epithelium of the neonatal rat and compared their time and site of appearance with those of basolateral markers, Na-K-ATPase and fodrin. The proteins studied were dipeptidyl peptidase IV (DPPIV) (microvilli), actin and villin (microvillar cytoskeletal proteins), glycoprotein 330 (gp330) (coated pits), and clathrin (coated pit cytoskeleton). Although apical microvilli and coated pits were first seen in the stage III nephron, many brush border markers including DPPIV, actin, and clathrin appeared earlier in the development and initially were not polarized. Only during stage III did they become concentrated at the apical membrane. Villin first appeared in the stage III proximal tubule where it was located diffusely in the cytoplasm and in lysosomes as well as along the apical membrane. It did not completely colocalize with actin until stage IV. Gp330 first appeared during stage III and from the beginning was restricted to the apical clathrin-coated membrane domains and endosomes. The results demonstrate that 1) the expression of renal brush border proteins during development is asynchronous, and 2) unlike the basolateral plasmalemmal domain, which is established early in nephrogenesis, brush border assembly occurs later, approximately coinciding with the onset of glomerular filtration.