The target site for cantharidin (CA) and its analogues was isolated recently from mouse liver and identified as protein phosphatase 2A (PP2A) in the heterodimeric form known as PP2A2. The most toxic CA analogue, endothall thioanhydride (ETA) (mouse i.p. LD50 0.3 mg/kg), appears to have the same binding site in mouse liver and brain based on studies comparing [H-3]ETA and [H-3]CA. ATP and its nonhydrolyzable analogues and pyrophosphate and related compounds including phosphonoformic acid inhibited both [H-3]CA and [H-3]ETA binding with IC50 values ranging from 2 to 81 muM. As with CA itself, the most potent inhibitors have two negatively charged groups in close proximity to each other. Inhibition of [H-3]CA binding by 5,5'-dithiobis(2-nitrobenzoic acid) and stimulation by N-ethylmaleimide indicated the involvement of a thiol site in the CA-binding domain. CA and three analogues (cantharidic acid, palasonin and endothall) inhibited PP2A and protein phosphatase 1 (PP1) but not PP2B or PP2C. The catalytic subunit of PP2A was 5- to 12-fold more sensitive to these CA analogues than the catalytic subunit of PP1. CA and the herbicide endothall also inhibited spinach leaf PP1 and PP2A and, at 50 muM, decreased the PP2A-mediated light-induced activation of nitrate reductase in intact spinach leaves by 62 and 56%, respectively. This is consistent with PP2A as their site of action in plants, and indicates the potential use of CA analogues as pharmacological probes to investigate cellular processes that are regulated by reversible protein phosphorylation in vivo.