Chiriquitoxin (CHTX, 1), isolated from the Costa Rican frog Atelops chiriquiensis, has drawn the interest of physiologists because of its effects on both Na and K currents of exitable cell membranes. However, the structure has remained undetermined. We isolated 6mg of CHTX from 100g of the frog skin and deduced the molecular formula C_<13>H_<20>N_<4>O_<10> from HRFABMS data. The positive reaction of CHTX to ninhydrine reagent indicated the presence of NH_2 in the molecule. The FT-IR and NMR spectra data together with H_5IO_6 oxdation experiments suggested that CHTX was a tetrodotoxin (TTX, 2) analog, in which 11-CH_2OH was replaced by CH(OH)CH(NH_2)COOH or CH(NH_2)CH(OH)COOH. In a D_2O solution containing 1%CF_3COOD and 4%CD_3COOD, CHTX formed a 13,6-lactone (4) of which CH(NH2) was exchanged with deuterium. Thus the substituent group at C-6 was presumed to be CH(OH)CH(NH_2)COOH. The stereochemistries of C-6 (S), C-11 (R), and C-12 (S) were suggested by NOE measurements and difference spectra.