The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutinin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the alpha-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5-8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13-21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean +/- SEM of 6.86 +/- 0.04 (n = 121) while the near-membrane pH of older cells was 6.61 +/- 0.04 (n = 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx.