DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs), small RNA molecules that are predicted to silence the expression of ~30% of protein-coding genes. DICER1 utilizes its RNAse IIIa and IIIb endonuclease domains to cleave premature miRNA stemloops in order to release the mature single-strand miRNA, which can be coded within either the 5’ (5p) or 3’ (3p) arms of the stemloop structure. The RNAse IIIa domain cleaves the 3p arm of pre-miRNAs whereas the RNAse IIIb domain catalyzes the cleavage of the 5p arm. Germ-line mutations in DICER1 have been identified in patients afflicted with pleiotropic tumour predisposition syndrome whose tumours and lesions include: pleuropulmonary blastoma, cystic nephroma, ovarian Sertoli-Leydig cell tumour, thyroid non-toxic goiter/cyst, Wilms tumour, pituitary blastoma, pineoblastoma, intraocular medulloepithelioma, medulloblastoma/infratentorial primitive neuroectodermal tumour, and seminomas.  The majority of families with pleiotropic tumour predisposition syndrome have frameshift or nonsense mutations in DICER1 while their tumours/lesions bear additional missense mutations in the RNAse IIIb domain. The genetic events with respect to DICER1 syndrome appear to follow a variation of Knudson’s two-hit hypothesis for tumour formation, where one allele is inactivated and the other allele bears a mutation in the RNAse IIIb domain should they occur in susceptible cell type at the right period during development. A robust assay to evaluate the consequences of DICER1 mutations on miRNA production has not been generated. Here I present the results of our DICER in vitro cleavage assay. These results may prove informative for predicting the consequences of DICER1 mutations on miRNA generation.