The kinetics of lactate dehydrogenase in situ were studied in sections of unfixed liver of the male mouse using a quantitative histochemical technique. The sections were incubated on substrate-containing gel films. The absorbance of the final reaction products deposited in a single hepatocyte was measured continuously during the incubation as a function of incubation time using a scanning microdensitometer. The absorbance increased non-linearly during the first minute of incubation, but linearly for at least the next 3 min afterwards. The initial velocity (vi) of the dehydrogenase was calculated from two equations proposed previously by us, vi = 2.82 degrees A and vi = vi + 2 degrees A, where vi and degrees A are, respectively, the gradient and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min. The dependence of vi on lactate concentration gave the following mean kinetic constants. For periportal hepatocytes, the apparent Km = 14 mM and Vmax = 80 mumoles hydrogen equivalents formed cm-3 hepatocyte cytoplasm min-1. For pericentral hepatocytes, Km = 12 mM and Vmax = 87 mumoles hydrogen equivalents cm-3 min-1. The Km values are very similar to those determined previously from biochemical assays. The concentrations of the enzyme in single hepatocytes calculated from the Vmax values are in good agreement with those obtained by another method. These data substantiate the validity of our equations.