A procedure for inducing and detecting the loss of conditionally dispensable (CD) chromosomes in filamentous fungi during vegetative growth was developed using Nectria haematococca mating population VI as a model. CD chromosomes in two different isolates of N. haematococca were tagged via integrative transformation with a gene conferring resistance to hygromycin B. In each case the transformation vector included chromosome-specific DNA in order to direct its homologous recombination with the desired chromosome. Chromosome loss was induced by exposing tagged isolates to inhibitory concentrations of benomyl either for protracted periods of time on solid medium or for short periods of time in liquid medium. After exposure to benomyl, isolates that lost the tagged chromosome were identified by their loss of resistance to hygromycin B. Electrophoretic karyotyping was used to verify that isolates which failed to grow on hygromycin B lacked an intact CD chromosome. Ten other chemicals known to interfere with mitotic events or cell development in other organisms did not induce CD chromosome loss in N. haematococca.