The purpose of this work was to determine if physiological variations in stromal ionic composition can affect light transmission independent of hydration in the isolated bovine corneal stroma. The hydration and spectral transmission of paired stromal strips were monitored over three hours in Krebs-bicarbonate Ringer solutions with high or low concentrations of either sodium chloride (110% vs 90%), potassium chloride, magnesium sulfate, or calcium chloride (200% vs 50%), and high vs low pH (7 vs 8). Wet tissue weights were determined immediately after spectral scans performed in cuvettes (% transmission vs wavelength over 380 to 750 nm). Following each series of scans, stromal strips were dried and weighed for hydration determinations. Hydration dynamics were found to be independent of variations in sodium, potassium, magnesium, pH, chloride and sulfate over the physiological range. Three-dimensional graphs of hydration vs light transmission vs light wavelength demonstrated that light transmission is proportional to the cube root of wavelength, and decreases with increasing hydration. With each ionic variation, the three-dimensional graph of hydration vs spectral transmission was superimposable, within 7% transmission, at each wavelength and hydration, on the graph obtained with normal Ringer solution. Thus we conclude that the three-dimensional relationship between stromal hydration, light transmission and wavelength is not significantly dissociated by ionic fluctuations within the physiological range.