Two seryl-tRNAs, which were prepared from natural suppressor tRNA-rich fractions on a pattern of chromatography on Sephadex A-50, were phosphorylated by a tRNA kinase in Escherichia coli B. A part of phosphate on the tRNA was confirmed to be phosphoserine. Phosphoseryl-tRNA is universal in bacteria and vertebrates. Phosphoseryl-tRNA should be an intermediate from seryl-tRNA to selenocysteyl-tRNA.