The present study characterizes the inhibitory effects of nodularin, a recently isolated hepatotoxic compound from the cyanobacterium Nodularia spumigena, on type 1 (PP1), type 2A, (PP2A), type 2B (PP2B), and type 2C (PP2C) protein phosphatases. Both PP2A and PP1 were potently inhibited (IC50 = 0.026 and 1.8 nM, respectively) by nodularin, whereas PP2B was inhibited to a lesser extent (IC50 = 8.7 microM). Nodularin had no apparent effect on PP2C, alkaline phosphatase, acid phosphatase, insulin receptor tyrosine kinase, protein kinase A, phosphorylase kinase, or protein kinase C. In a whole-cell extract of T51B liver cells, nodularin inhibited PP1 and PP2A activity with a potency similar to that seen with their purified catalytic subunits. Thus, due to the high specificity of nodularin for PP2A and PP1, this hepatotoxin may prove to be useful as a probe for distinguishing the activity of these protein phosphatases in cell extracts.