Tautomycin inhibited the catalytic subunits of protein phosphatase‐1 (K _{i app} = 0.16 nM) more potently than protein phosphatase 2A (K _{i app} = 0.4 nM), and the native forms of these enzymes in mammalian, protozoan and plant extracts were inhibited in a similar manner. Protein phosphatase 2B was inhibited 10 000‐fold less potently, while two other phosphatases and six protein kinases were unaffected at 10 μM. Okadaic acid prevented the binding of tautomycin to protein phosphatase 2A, indicating a common binding site for both inhibitors. The different relative potencies of tautomycin and okadaic acid for protein phosphatases 1 and 2A suggest that parallel use of both inhibitors may help to identify physiological substrates for each enzyme.