The catalytic subunit of protein phosphatase 2A (PP2A c ) was purified from Neurospora crassa extract by (NH 4 ) 2 SO 4 -ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M r of PP2A c was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2A c was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the α-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2A c is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2A c did not cross-react with N. crassa PP2A c , indicating sequence differences outside the catalytic core of the enzyme.