Previous studies have shown that insulin, insulin-like growth factors, and interferon-gamma cause an increase the paracellular permeability across T84 human colonic epithelial cell monolayers. The striking similarity in the time course and the structural changes in the actin cytoskeleton prompted us to test whether these factors influenced the paracellular permeability by a common mechanism. T84 cell monolayers were grown in variously modified media, and the effect of all three factors was tested by measuring the conductance of monolayers mounted in Ussing chambers. Media additions or deletions that modify cellular carbohydrate metabolism markedly influenced the response to all these peptides. In particular, the increase in conductance produced by each of the three peptides was 1) inhibited by addition of short-chain fatty acids, principally n-butyrate and propionate; 2) attenuated by the replacement of media glucose with slowly metabolized and nonmetabolized sugars; 3) potentiated by decreasing the media iron concentration; and 4) inhibited by increasing the media iron concentration. The effects of short-chain fatty acids, glucose, and iron were all shown to be dose dependent. In addition, the ability of high media iron to prevent the increase in permeability caused by all three factors was shown to require ambient oxygen. Together, these results strongly suggest that insulin, insulin-like growth factors, and interferon-gamma increase the permeability across T84 cell monolayers through a common mechanism that is modulated by glucose-derived metabolites and oxidative metabolism.