Random insertional mutagenesis of Colletotrichum higginsianum using Agrobacterium tumefaciens-mediated transformation was utilised to tag genes required for fungal pathogenicity. Two of the mutants, path-19 and path-35, were arginine auxotrophs and produced no symptoms on susceptible Arabidopsis thaliana plants. The T-DNA tagged genes showed high homology to the polyprotein arg-6 precursor and carbamoyl-phosphate synthetase, respectively, both of which are involved in arginine biosynthesis. In both mutants, conidial germination and appressorium formation were similar to the wild-type strain, but appressorial penetration and subsequent invasive growth in planta were severely reduced. Complementation of the T-DNA tagged mutants with cosmid genomic clones containing wild-type copies of the respective genes fully restored pathogenicity and the ability to grow on minimal media. These results suggest that arginine biosynthesis is critical for the early stages of plant infection by C. higginsianum.