In yeast, many proteins are found in both the cytoplasmic and extracellu- lar compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100mM dithiothreitol increased the reac- tion rate, consistent with increased access of the enzyme after reduction of cell wall di- sulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine- specific membrane-impermeant biotinylation reagent specifically inactivated extracellu- lar GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4� 104 active GAPDH molecules per cell at steady state, and secre- tion studies showed replenishment to that level 1 h after inactivation. These results es- tablish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.