The micronutrient selenium is found in proteins as selenocysteine (Sec), the 21st amino acid cotranslationally inserted in response to a UGA codon. In vitro studies in archaea and mouse showed that Sec-tRNA Sec formation is a 3-step process starting with serylation of tRNA Sec by seryl-tRNA synthetase (SerRS), phosphorylation of serine to form phosphoserine (Sep)-tRNA Sec by phosphoseryl-tRNA Sec kinase (PSTK), and conversion to Sec-tRNA Sec by Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic selenoprotein synthesis has been lacking. Here, we present an analysis of Sec-tRNA Sec formation in the parasitic protozoon Trypanosoma brucei in vivo. Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis, demonstrating the essentiality of both enzymes for Sec-tRNA Sec formation. Growth of the 2 knockout strains was not impaired; thus, unlike mammals, trypanosomes do not require selenoproteins for viability. Analysis of conditional RNAi strains showed that SerRS, selenophosphate synthase, and the Sec-specific elongation factor, EFSec, are also essential for selenoprotein synthesis. These results with T. brucei imply that eukaryotes have a single pathway of Sec-tRNA Sec synthesis that requires Sep-tRNA Sec as an intermediate.