The availability of a large number of gene-disrupted mutants (either from natural mutants' collections or from knockout projects) is a great advantage for functional analysis studies. However, disfunction of many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes in cell morphology, conferring a major difficulty in studying null mutants. Therefore, obtaining variable levels of reduction in gene expression, especially of essential genes or genes whose impaired expression confers a pleiotropic phenotype, is extremely beneficial for studying their function. Here, we describe the use of RNAi as a gene silencing mechanism, in a manner that might facilitate the functional analysis of such essential genes. Two alternative strategies for the construction of an RNAi-induced inverted-repeat construct are demonstrated and a third alternative is suggested. In addition, DNA-mediated transformation of conidia by electroporation, RNA extraction from fungal mycelium and northern blot analysis are described in detail.The experimental results presented, demonstrate that RNAi can be employed as a gene silencing tool in Neurospora crassa, both for nonessential (al-2) and essential (cot-1) genes, resulting in a range of stable, partially silenced mutants, exhibiting different phenotypes.