Abstract: We describe a novel methodology for the comparison of protein samples called differential gel exposure (DifExpo). This method is based on the coelectrophoresis on a two-dimensional (2-D) gel of two protein samples. The samples are differentiated from each other by in vivo radiolabelling, using (14)C- and (3)H-isotopes. After 2-D separation and transfer on a polyvinylidene difluoride membrane, the (3)H/(14)C ratio of each protein spot is determined by exposure to two types of imaging plates, one sensitive to (14)C and the other to both (14)C and (3)H. We showed that DifExpo allows us to compare the cellular levels of several hundred proteins of the yeast proteome. Its sensitivity is comparable to silver staining. We also showed that it can be used to investigate changes in the rate of synthesis of individual proteins.