The amino acid selenocysteine is encoded by the triplet UGA. In bacteria, the differentiation between a UGA stop codon and a selenocysteine codon is mediated by a downstream mRNA secondary structure (SECIS, selenocysteine insertion sequence), which is recognized by the selenocysteine-specific elongation factor SelB. Compared to other bacteria, the Gram-positive anaerobe Eubacterium acidaminophilum shows an unusual high number of selenoproteins. Up to now, eight different selenocysteine-containing polypeptides have been identified. Secondary structures resembling the SECIS element of fdhF-mRNA from E. coli were postulated to exist downstream of the UGA codon in the corresponding mRNAs. However, these structures apparently did not show any homology at the sequence level. To establish the presence of these structures and their function regarding the incorporation of selenocysteine, the interaction between mRNAs and SelB from E. acidaminophilum was studied by gelshift experiments. However, a specific binding could not be observed. The gene of the selenocysteine-specific elongation factor from E. acidaminophilum could complement the selB-lesion of an E. coli mutant and promoted in vivo the incorporation of selenocysteine into fdhF-encoded formate dehydrogenase H. Obviously, the protein recognizes the SECIS-Element of fdhF-mRNA. The expression of the selenoprotein genes grdB (glycine reductase, protein B), prxU (peroxiredoxin) und selD (selenophosphate synthetase) from E. acidaminophilum in E. coli was studied by labeling the polypeptides with 75Se. The heterologously synthesizedproteins did not contain selenium. Thus, E. coli SelB did not recognize the secondary structures of the mRNAs as SECIS elements. However, coexpression of selB from E. acidaminophilum resulted in the incorporation of selenocysteine into the corresponding gene products. Using this expression system the function of the postulated mRNA secondary structures was proven by introducing several mutations. These alterations significantly influenced the selenium content of the proteins. Translational fusions containing the SECIS elements (including the selenocysteine codon) of grdB, prxU and selD between gst and lacZ were constructed. Efficient UGA decoding was observed in the presence of selB from E. acidaminophilum. The amount of full length fusion protein reached up to 65 % compared to the corresponding cysteine mutants (containing a UGC triplet in place of UGA).