A new enzyme (isobutylidenediurea amidinohydrolase) catalyzing the hydrolysis of isobutylidenediurea (a condensation product of urea and isobutyraldehyde widely used as a slow-release nitrogeneous fertilizer) was characterized from a strain of Rhodococcus erythropolis. The enzyme was purified 1,250-fold to apparent homogeneity and shown to hydrolyze the fertilizer to urea and isobutyraldehyde at a molar ratio of 2: 1. No activity was observed with ureido- or other structurally related compounds. Its molecular mass was determined by native polyacrylamide gelelectrophoresis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry to be 15 kDa (+/-2 kDa) and 16.4 kDa, respectively. Growth of the bacterium in the presence of isobutylidenediurea led to an increased expression of the constitutively synthetized enzyme.