The ability to produce melanin is a key virulence factor in many fungal pathogens including the human basidiomycete pathogen Cryptococcus neoformans, a major cause of life-threatening infections among immunocompromised persons. Despite the significance of melanin biosynthesis in virulence of C. neoformans, the cellular and molecular processes involved in this pathway have not yet been fully elucidated. Here, we used Agrobacterium to isolate insertional mutants and screened 12 000 mutants to uncover genes involved in melanin production in C. neoformans. Four new mutant alleles of the well-known melanin biosynthesis gene, LAC1, which encodes laccase were identified, and the T-DNA was shown to have a possible predisposition for insertion into the promoters of genes, in particular LAC1. Melanization in C. neoformans is dependent on five additional genes identified in this screen encoding homologues of the copper transporter Ccc2, the copper chaperone Atx1, the chitin synthase Chs3, the transcriptional coactivator Mbf1 and the chromatin-remodelling enzyme Snf5. Illumination of the molecular and genetic components of this virulence pathway reveals potential novel targets for drug development against C. neoformans and provides further insight into the intimate relationship between metal ion homeostasis and melanin biosynthesis.