Diguanidinobutanase (EC 3.5.3.20), which catalyses the hydrolysis of 1,4-diguanidinobutane (DGB) to agmatine (1-amino-4-guanidinobutane) and urea, was purified to homogeneity from Pseudomonas purida ATCC 12633, The enzyme had a molecular mass of 170 kDa and was suggested to be a tetramer of subunits that had a molecular mass of 38 kDa. The enzyme contained two Mn2+ ions per subunit. DGB was the most effective substrate and its K-m, was 0.65 mM. The turnover number for the subunit at saturation with DGB was 1330 molecules s(-1). The higher homologues of DGB with five to seven methylene groups were also hydrolysed effectively. Agmatine was hydrolysed at a rate of 0.6% of that observed with DGB. The agmatine homologues with five to seven methylene groups were hydrolysed, although the rates were low. The enzyme was sensitive to p-chloromercuribenzoate. Agmatine sulphate was enzymatically prepared from DGB. The purified product, free from detectable putrescine and DGB, was obtained with a yield of 93% (mol/mol).