We report evidence of a factor secreted at the apical side of epithelial monolayers which modulates tight junction structure and permeability. This activity was detected within 4-7 days of conditioning of the apical medium by MDCK, A6 or Caco-2 epithelial cell lines cultured on permeable membranes in bipartite chambers. Apical conditioned medium (ACM), applied to the basolateral surface of a confluent monolayer, increased the transepithelial electrical resistance (TER), progressively reaching values 12-22% higher than the baseline within 5-10 min. After 40-60 min, the TER returned slowly to the basal value. This phenomenon was not observed either when using preheated ACM or the ACM filtrate obtained through a 30,000 MW cutoff membrane. The ACM maintained its activity even when applied to cell lines from different organs and species, as demonstrated when ACM from MDCK monolayers promoted an increase of 22% in the TER of Caco-2 cells. The increase of TER induced by the ACM treatment is accompanied by a change in the distribution of the number of tight junction strands, from an initial pattern, dominated mostly by junctions with one or two strands, to a new pattern after treatment dominated by junctions with two or three strands. Our results suggest the existence of a mechanism in epithelial cells that could signal leakage of apically secreted components to the basolateral side, thereby modulating the junction structure and permeability.