The mechanism of selenocysteine insertion into proteins is distinct from all other amino acids in all lines of descent in that it needs specific protein cofactors and a structurally unique tRNA(Sec). It is first aminoacylated with serine and further recognized among all other serylated serine isoacceptors by a selenocysteine synthase and is converted to selenocysteyl-tRNA(Sec). We present here the complete set of identity elements for selenylation of mammalian seryl-tRNA(Sec) and show that the transplantation of these elements into normal serine tRNA allows its selenylation. Four particular structural motifs differentiate eukaryotic tRNA(Sec) from normal tRNA(Ser): the orientation of the extra arm, the short 4 bp T psi C-stem, the extra long 9 bp acceptor-stem and the elongated 6 bp dihydrouridine-stem. Only the last two are essential and only together sufficient for selenocysteine synthesis, whereby the additional base-pairs of the acceptor-stem may be replaced by non-paired nucleotides. Each exchange of the first three structural motifs mentioned above between tRNA(Ser) and tRNA(Sec) resulted in a significant loss of serylation, indicating that the overall composition of particular structure elements is necessary to maintain normal functions of tRNA(Sec). Since we find that all seryl-tRNAs which are selenylated are also substrates for serine phosphorylation we propose that phosphoseryl-tRNA(Sec) is a storage form of seryl-tRNA(Sec).