The gene encoding edeine B₁ amidinohydrolase from Bacillus brevis TT02-8 was cloned into Escherichia coli and its nucleotide sequence was determined. An open reading frame was identified and was found to encode a polypeptide of 289 amino acid residues with a predicted molecular weight of 32, 455, which was consistent with that previously calculated for edeine B₁ amidinohydrolase purified from this bacterium. Comparison of the deduced amino acid sequence of this enzyme with other amidinohydrolases revealed the highest homology to B. subtilis agmatine ureohydolase. The enzymatic activity of the protein produced in Escherichia coli was analyzed. Three histidine residues, H-112, H-137 and H-151 in the edeine B₁ amidinohydrolase, which are highly conserved in amidinohydrolases, were changed to alanine by site-directed mutagenesis. Analysis of each of these mutants revealed that three histidine residues are important but not essential for the enzyme activity.