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Previous studies have indicated that detoxification of their hosts’ phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and virulence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates of Pythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts’ phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized enzyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cytochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating population VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share limited nucleotide similarity with N. haematococca PDA genes.