Reverse-phase high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used to identify Pacific ciguatoxins (P-CTX) and P-CTX congeners present in a highly purified extract from the viscera of ciguateric moray eels (Lycodontis javanicus) collected in the central Pacific Ocean. Fourteen P-CTX or P-CTX congeners were identified with protonated molecular ions [M + H]+ m/z 1095.7 (two), 1111.6 (six) or 1127.7 (six), including dominant ions for P-CTX-1, -2 and -3. In addition to the protonated species, each of these ciguatoxins gave rise to prominent [M + NH4]+ and [M + Na]+ ions. The 11 new P-CTX congeners, not readily detected by mouse bioassay, were present in trace amounts (2-13% of P-CTX-2 levels) and identified as several oxidized P-CTX-1, -2 and -3, and a possible diasteriomer of P-CTX-1. Acetonitrile-water gradients buffered with 1 mM ammonium acetate improved the separation and detection of the minor ciguatoxins compared with an acetonitrile-water gradient modified with 0.1% TFA. Turbo-assisted HPLC/MS had sufficient sensitivity to detect P-CTX-1 in a crude extract from the flesh of an Australian ciguateric fish. Compounds with masses equivalent to other isolated ciguatoxins, including Caribbean-CTX-1, gambiertoxin-4A and P-CTX-3C, were not detected in these samples. HPLC/MS can readily identify multiple ciguatoxins accumulated by fish and has the potential to be used as a confirmatory analytical method for characterizing the low levels of ciguatoxins contaminating ciguateric fish.