The continuous cultivation technique has been used to screen for microorganisms producing D-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5’-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l(-1) in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the D-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.