The effect of a series of phytotoxins isolated from the fungus Guanomyces polytrix on calmodulin (CaM)-dependent nicotinamide adenine dinucleotide kinase (NADK) and CaM-dependent cyclic nucleotide phosphodiesterase (PDE) activities was investigated. The results indicated that (2S,3S)-5-hydroxy-6,8-dimethoxy-2,3-dimethyl-4H-2,3-dihydronaphtho[2,3-b]-pyran-4-one, (2S,3S)-5-hydroxy-6,8,10-trimethoxy-2,3-dimethyl-4H-2,3-dihydro-naphtho[2,3-b]-pyran-4-one, (2S,3R)-5-hydroxy-6,8-dimethoxy-2,3-dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one, (2S,3R)-5-hydroxy-6,8,10-trime-thoxy-2,3-dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one, 5-hydro-xy-6,8-dimethoxy-2,3-dimethyl-4H-naphtho[2,3-b]-pyran-4-one, rubrofusarin B, and ergosta-4,6,8(14),22-tetraen-3-one inhibited the activation of both target enzymes in the presence of CaM. On the other hand, (2S)-5-hydroxy-6,8-dimethoxy-2-methyl-4H-2,3-dihydronaphtho[2,3-b]-pyran-4-one and (2S)-5-hydroxy-6,8,10-trimethoxy-2-methyl-4H-2,3-dihydronaphtho-[2,3-b]-pyran-4-one inhibited the activation of PDE and the basal activity of NADK. Thus, these phytotoxins are CaM inhibitors and may exert their phytotoxic action by inhibiting the CaM-dependent process, although they could also interfere with other cellular metabolic phenomena. This is the first report of the use of the NADK assay to detect or quantify CaM inhibitors, and it could be a valuable tool for studying those CaM isoforms regulating NADK.