By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.