Two forms of NAD+-isocitrate dehydrogenase, named ICDH-1 and ICDH-2, have been identified and purified in Phycomyces blakesleeanus NRRL-1555(-). These enzymes forms may be separated by chromatography on DEAE-Sephacel. ICDH-2 induction was a response to the adaptation of Phycomyes growth on acetate as the carbon source. Both enzyme forms were octamers of 388 + or - 30 kDa with apparently identical subunits of 40.5 +/- 5 kDa, but they were distinguishable by their electrophoretic mobilities on polyacrylamide gel electrophoresis. Isoelectric pH values were 5.28 and 4.96 for ICDH-1 and ICDH-2, respectively. ICDH-2 was more stable to urea denaturation than ICDH-1. At pH 7.6, ICDH-1 showed a markedly sigmoidal kinetic behavior with respect to isocitrate. However, ICDH-1 and ICDH-2 showed hyperbolic kinetics with respect to NAD+. THe tribasic form of isocitrate (I3-) and its magnesium complex (MI-) are the true substrates for both enzyme forms. Kinetic data obtained with Mg2+ as a divalent cation for both enzyme forms are compatible with the kinetic mechanism proposed by Cohen and Colman (1974) [Eur. J. Biochem. 47, 35-45] but assuming some degree of interaction between binding sites for the active form of isocitrate. This report describes for the first time the existence of two forms of NAD+-isocitrate dehydrogenase in filamentous fungi. From the changes in activity levels for each form, during adaptation of Phycomyces to growth on acetate and taking into account the kinetic and regulatory properties of both enzyme forms, we discuss the role of ICDH-1 and ICDH-2 in the control of isocitrate flux in Phycomyces.