Electron microprobe analysis was employed to measure the intracellular electrolyte concentrations of the bullfrog corneal epithelium. Under control conditions, transepithelial potential short-circuited and both sides of the isolated cornea incubated in Conway's solution, the mean intracellular concentrations were 8.0 for sodium, 18.4 for chloride and 117.3 for potassium (mmole/kg wet weight). These values are in good agreement with previously reported ion activities implying that the intracellular activity coefficient for small ions is close to that of the extracellular space. No significant differences between the nuclear and cytoplasmic concentrations of small diffusible ions were detectable. Similarly, the chloride concentrations in the different epithelial layers were virtually identical and showed parallel changes at varying states of chloride secretion, suggesting that corneal epithelium represents a functional syncytium. The behavior of the intracellular ion concentrations after removal of sodium, chloride or potassium from the outer or inner bath is consistent with a passive electrodiffusive efflux of chloride across the outer membrane and a sodium coupled chloride uptake across the inner membrane. Furthermore, effects of bicarbonate and CO on the rate of chloride secretion and on the intracellular sodium and chloride concentrations were observed, indicating a possible role of pH in regulating chloride secretion.