Variations in the volume and width of lateral intercellular spaces (LICS) of dog tracheal mucosa in vitro were investigated by use of stereological and linear measurements of electron micrographs. Alterations in the volume or width of LICS were then correlated with physiological conditions and electrical parameters. LICS were quite narrow between the ciliated cells compared with those around the nonciliated dark cells (goblet, brush, and basal cells). LICS comprised 6.8 +/- 2.9% of tissue volume in preparations that were mounted in an Ussing chamber and short-circuited, whereas in unmounted and open-circuited tissues it occupied only 1 +/- 0.2% of the volume of the preparations (P less than 0.016, n = 5). The effects of stimulation of Cl secretion by 1 microM epinephrine were tested. In seven epinephrine-treated tissues LICS volume was 2.9 +/- 0.9% of total epithelial volume compared with 8.7 +/- 2.9% in control tissues (P less than 0.015). The width of LICS around dark cells in epinephrine-treated tissues was 0.42 +/- 0.06 micron compared with 0.98 +/- 0.13 micron in control tissues (P less than 0.001). The data suggest that LICS act as pliable fluid reservoirs that empty and collapse on stimulation of Cl secretion.