The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3¢-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mMKCl) anddependedon thepresenceof 5 mMMgCl2. In vitrosynthesizedU6 snRNA without a 3¢-terminal UMP residue was not accepted as substrate.