The ability of the fungus Nectria haematococca to demethylate pisatin is required for its tolerance to this phytoalexin and for its virulence on Pisum sativum. Pisatin demethylase activity (Pda) is inducible by pisatin, and among Pda+ N. haematococca isolates there are large differences in the level of activity observed after induction. Previous analysis of two field isolates identified three genes controlling pisatin demethylation. Pda1 confers a high level of demethylase activity (PdaSH phenotype), while Pda2 and Pda3 encode low rates of demethylation (PdaLL). High virulence on pea was associated only with Pda1. The present study was an analysis of the control of pisatin demthylation in field isolate T-23, which expressed an intermediate level of activity (PdaSM), and in another PdaLL field isolate, T-161. A fourth gene, Pda4, was found to control the PdaSM phenotype, while the Pda gene in isolate T-161 may represent another allele at the Pda3 locus. In crosses segregating for Pda4 or Pda3, only progeny with the PdaSM phenotype were virulent; both PdaLL and Pda- progeny were nonvirulent. In a cross involving Pda1 and Pda4, PdaSH progeny were more virulent than PdaSM progeny. Apparently the amount of phytoalexin-detoxifying enzyme produced by this fungus can directly affect its virulence on pea.