The spatial and temporal relationships between cytoplasmic filaments and the morphogenesis of the intestinal brush border were examined by transmission electron microscopy of normally developing tissue and of tissue exposed to a variety of experimental conditions in organ culture. Distinct stages in the development of the brush border were identified: (1) Irregular projections of the apical plasma membrane that contain a network of microfilaments are converted to uniform projections filled with a core bundle of straight microfilaments (7-11d of incubation). (2) Rootlets form by an elongation or aggregation of filaments (11-15d). (3) The terminal web forms first as a network of short filaments just below the apical plasma membrane, then secondarily stratifies into two layers (19d of incubation to 3d posthatching). (4) Core filaments elongate as microvilli achieve their maturity (21d of incubation to 5d posthatching). Microvillus formation was not perturbed by culturing 9d tissue in high concentrations of Ca++ or Mg++, either with or without the ionophore, A23187. Rootlet formation was stimulated by high Mg++, with or without A23187, and, for reasons unknown, by ethanol. Terminal web formation was not stimulated by Mg++ or Ca++, but the integrity of the terminal web was lost when 21d embryonic tissue was cultured with EGTA or cytochalasin B. After stratification, the terminal web could not be disrupted by EGTA, but instead was aggregated to the center of the apical end of the cell.