Phosphatidate phosphatase (EC 3.1.3.4) was detected in filamentous pathogenic fungi. In both dermatophytes,Microsporum gypseum andEpidermophyton floccosum, the enzyme was located in the mitochondrial and microsomal subcellular fractions with a pH optimum of 6.0. TheE. floccosum enzyme was more active than that ofM. gypseum. Although the enzyme in both fractions of the dermatophytes was susceptible to inhibition by Fe(2+), Mn(2+), Cu(2+), Ba(2+) and Hg(2+), theE. floccosum microsomal enzyme was completely inhibited by Mn(2+) and Fe(2+). The enzyme ofM. gypseum as well as ofE. floccosum exhibited a Mg(2+) dependency in presence of EDTA. Sulphydryl reagents did not inhibit either the mitochondrial or the microsomal enzyme of these fungi. Phosphorylcholine and lysophosphatidylcholine markedly inhibited the enzyme in the two dermatophytes; however, the inhibition was less pronounced with phosphatidylcholine.