A combination of high specific activity [14C]glucose, two-dimensional chromatography, and autoradiography can be utilized to follow the flow of glucose carbon into lactose and various intermediary metabolites of the mammary gland. The technique described requires a minimum amount of tissue manipulation, is quantitative, utilizes microgram quantities of sample, and can be used for tissue pieces, single cell suspensions, and cells in culture. Clean separation of lactose from intermediates of glucose metabolism in a single chromatogram will aid the study of the regulation of mammary-specific functions. © 1979.