Selenophosphate synthetase (SelD) generates the selenium donor for 
selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes 
is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as 
a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, 
synthesized selenophosphate from selenide, whereas SPS1 may utilize a different 
substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain 
unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells 
using small interfering RNA and found that selenoprotein biosynthesis was 
severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 
into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, 
indicating that SPS1 cannot complement SPS2 function. These in vivo studies 
indicate that SPS2 is essential for generating the selenium donor for 
selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more 
specialized, non-essential role in selenoprotein metabolism.