Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway are excellent recipient strains for gene targeting approaches. In addition, NHEJ-deficiency can facilitate the formation of heterokaryons which allows rapid identification of essential genes. However, the use of NHEJ-deficient strains can also pose some limitations for gene function analyses. For example, lack of the NHEJ pathway can interfere with phenotypic analyses and complicate complementation studies. Moreover, heterokaryons are difficult to propagate and re-transform. We describe here strategies and methods to circumvent these problems and to better exploit the power of NHEJ-deficient strains. We provide methods for the establishment of transiently deficient NHEJ strains, for improved complementation analyses using AMA1-based vectors and for fast identification and propagation of heterokaryons. The methods described are applicable for a wide range of filamentous fungi.