A method is described for rapidly quenching metabolism in yeast and extracting metabolites for analysis. The saponin digitonin is used to permeabilize the yeast cell membrane, in conjunction with perchloric acid (PCA) to quench metabolism and extract metabolites. Using this digitonin-PCA quench and extraction procedure, we have determined ATP concentrations in Saccharomyces cerevisiae in the range 2.89-3.39 mM, [ATP]/[ADP] ratios of 4.5-6.6, and phosphorylation potentials of 48.7-49.9 kJ.mol-1. A direct comparison with the currently accepted freeze/grind PCA/thaw procedure for extraction of yeast cell metabolites shows that essentially the same values are obtained by both techniques. The digitonin-PCA quench extraction method, used in conjunction with automated enzymatic analyses of metabolites, allows rapid extraction and analysis of large numbers of samples and metabolites and hence permits detailed investigations of intermediary metabolism in yeast.