The beta-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes. Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission. We analyzed three fluorogenic substrates (FDG, C12FDG, and CMFDG), all of which incorporate fluorescein as their fluorophore, regarding intensity of fluorescent signal and selectivity toward the transfected beta-galactosidase activity vs. lysosomal enzyme activity. Among these substrates, 5-chloromethylfluoresecein di-beta-D-galactopyranoside (CMFDG) showed the strongest selectivity toward the lacZ-encoded enzyme activity. An attempt to improve this selectivity by alkalinization of the lysosomal pH with chloroquine, such that the endogenous enzyme would be exposed to a suboptimal pH, led to significant cell death. In contrast, inhibition of dye efflux with verapamil enhanced the selectivity of CMFDG toward the lacZ-encoded enzyme activity by approximately threefold. Incubation with probenecid, on the other hand, showed little effect.