In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB, the helicase uvrD1 and a double knock-out lacking both proteins were constructed and their sensitivity to a series of DNA damaging agents wa analysed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and inter-strand cross-links. In addition, it could be shown to exert a protective effect against oxidising and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria, which lack the postreplicative mismatch repair system, NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.