[3H]Puromycin was covalently incorporated into rat liver ribosomes and isolated 40S and 60S subunits on irradiation at 254 nm. A study of the concentration dependence of this photolytic incorporation suggested that it arose from specific sites on isolated subunits but also from unspecific ones in the case of ribosomes, these sites being probably located on contaminant nonribosomal proteins. Puromycin was incorporated simultaneously into ribosomal proteins and rRNAs. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60S subunits and in 80S ribosomes, L10 being the most radioactive protein. Some antibiotics, which act on the peptidyltransferase center (amicetin and gougerotin), and also tetracycline competed with this labeling. Therefore, it was concluded that puromycin interaction with protein L10 occurred most likely at a functional site. In the case of free 40S subunits, labeling distribution among proteins was much wider. The possibility that proteins S3 and perhaps S23-24, which were significantly labeled in crude ribosomes too, also belong to a specific site interacting with puromycin is discussed.