Brevetoxins are natural neurotoxins that are produced by “red tide” algae. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps, samples were analyzed by liquid chromatography-electrospray mass spectrometry (LC-ES-MS). Two metabolites were found for brevetoxin-1: brevetoxin-1-M1 (MW 900 Da), formed by converting one double bond in the E or F ring into a diol; and brevetoxin-1-M2 (MW 884 Da), a hydrolysis product of brevetoxin-1 involving opening of the lactone ring. The incubation study of brevetoxin-2 found two metabolites. Brevetoxin-2-M1 (MW 912 Da) was elucidated by negative mode LC-MS/MS to be the hydrolysis product of brevetoxin-2. The second metabolite (brevetoxin-2-M2, MW 896 Da) was deduced to be brevetoxin-3. All brevetoxins have high affinities for sodium ions. Attempts to obtain informative product ions from the collision induced decomposition (CID) of [M + Na]+ brevetoxin precursor ions only resulted in uninformative sodium ion signals. In our nano-electrospray experiments, the addition of ammonium fluoride resulted in the formation of the ammonium adduct or protonated brevetoxin with a concomitant decrease of the sodium adduct peak. Product ion spectra of [M + NH4]+ and [M + H]+ were similar and provided useful structural information. The optimal values for ammonium fluoride concentration and the cone voltage were experimentally determined. In negative mode electrospray, without additives, deprotonated molecules of brevetoxins do not appear in high abundances, and thus are not well-suited for CID experiments. Several anions were tested for their abilities to form brevetoxin-anion adducts by mixing ammonium salts of these anions with brevetoxin-2 and brevetoxin-3. Under CID, [M + Cl]-, [M + Br]-, [M + OAc]-, [M + HCOO]-, [M + NO3]- adducts all produced only the respective anions in CID experiments, and thus, gave no structural information. In contrast, upon CID, both [M + F]- and [M + HCO3]- precursor adducts gave structurally-informative fragment peaks that exhibited similarities to those of [M - H]- ions; the detailed fragmentation mechanisms are discussed. In comparison, fluoride is a better choice to study brevetoxins in negative ES-MS by the anionic adduct approach.