Permeabilized intestinal absorptive cell brush borders contain a perijunctional ring of actin and myosin (PAMR) that can be induced to contract. Recently, morphological changes suggestive of PAMR contraction were shown to occur in absorptive cells of ileal epithelium after exposure to cytochalasin D (CD) (J. Cell Biol. 102: 2125-2136, 1986). With this response, altered tight junction structure and enhanced tight junction permeability also occur. To further assess the relationship between PAMR contraction and enhanced tight junction permeability, we examined the effect of the uncoupler 2,4-dinitrophenol (DNP) on this CD response. Progressive depletion of functionally defined intraepithelial energy stores occurred with DNP concentrations of 0.1-1 mM. Such DNP concentrations did not independently impair tight junction barrier function. Depletion of energy stores before CD exposure ablated the ability of CD to induce abnormalities of tight junction permeability. Similarly, PAMR condensation and alterations in tight junction structure could be dissociated from CD exposure by prior depletion of functional energy reserves. These data tie CD elicited alterations in tight junction structure and permeability to an energy dependent event that appears to be PAMR contraction. We speculate that tensile forces within the PAMR regulate tight junction structure and function.