A quantitative histochemical method to determine the apparent Km and Vmax values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025-1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at lambda 550 nm. The ratio between the absorbance at lambda 550 and lambda 500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 micrometers both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26 +/- 0.081 mM (weighted regression analysis) and 0.28-0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme. The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 micrometers, incubation time 3 min, 37 degrees C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data +/- SD from the jejunal villi read as follows. Apical: Km = 0.81 +/- 0.43 mM, Vmax = 3.99 +/- 1.217 absorbance units (A) and Km = 0.87 +/- 0.428 mM, Vmax = 4.02 +/- 1.191 A, respectively. Basal: Km = 0.82 +/- 0.261 mM, Vmax = 3.26 +/- 0.719 A and Km = 0.77 +/- 0.184 mM, Vmax = 3.04 +/- 0.518 AU, respectively. As demonstrated by factorial analysis of variance only Vmax is influenced by the villus position.