A one-pot synthesis of fluorescent bimetallic silver-gold nanoparticles in aqueous medium is presented. Carboxylic acid-functionalized nanoparticles were prepared with different metal compositions from 90:10 to 10:90 (n:n) for silver:gold with a diameter of 1.8±0.4 nm. Pure silver and gold nanoparticles were prepared for comparison. Spectroscopic analyses showed that the ligand, i.e. 11-mercaptoundecanoic acid, bind to the particle surface by the thiol group, leaving the carboxylic acid accessible for further functionalization, e.g. by suitable coupling reactions. Nanoparticles with a silver content up to 60:40 showed autofluorescence with a large Stokes shift of about 250-300 nm (maximum wavelength of the emission between 608 nm and 645 nm). The intracellular localization of bimetallic silver-gold nanoparticles was studied in HeLa cells by confocal laser scanning microscopy (CLSM). The alloyed silver-gold nanoparticles showed no significant cytotoxicity at a metal concentration of 5 µg mL-1 for 24 h, but were cytotoxic to some degree at 50 µg mL-1 at higher silver content.