In this paper, we report a simultaneous determination of azoxyglycosides by high performance liquid chromatography (HPLC) together with the variation of their contents in growing cycad leaves. An exact and rapid procedure is necessary to analyze hydrolysis and transglycosylation of azoxyglycosides catalyzed by β-glycosidase, and the analytical results are useful for studies on the recent poisoning of cattle caused by ingestion of cycad leaves in the southwestern islands of Japan. Specimens of cycasin, macrozamin, and neocycasin A, B, and C were isolated in our laboratory. Cycad leaflets were collected periodically from the leaves shooting on June 1981 of the same single trunk in the campus garden. Azoxyglycosides were extracted with 70% ethanol from 0.1 to 2 g of leaflets at low temperature. The extract was diluted 4 times with water and 10 μl of an aqueous solution of sulfosalicylic acid (250 mg/ml) was further added. After standing for 10 min at 0°C, the sample was passed through a membrane filter (0.2μm, FR-20, Fuji Photo Film Co. Ltd.). A portion (10 to 30μl) of the filtrate was applied to the column for chromatography. The instrument was a Trirotar high pressure liquid chromatograph equipped with a Uvidec-100-ll flow analyzer (Japan Spectroscopic Co. Ltd.). The column used was a packed one, S-614 from Showa Denko Co. Ltd., and aqueous methanol (85%, v/v) was used as the eluting solvent at a flow rate of 0.8ml/min. Absorption of the effluent was recorded at 215 nm (the maximum wavelength of azoxyglycosides), and the amount of each glycoside was calculated from the peak area. Azoxyglycosides were satisfactorily separated by HPLC. Neocycasin A, B, C, and macrozamin are β-laminaribioside, β-gentiobioside, β-laminari-tetraoside, and β-primeveroside of methylazoxymethanol (MAM), respectively. Analysis of other minor glycosides (neocycasin D, β-laminaritrioside; neocycasin E, β-cellobioside) was not attempted as their contents were negligible (below the detection limit) in cycad leaves. Macrozamin was found only at the young stage, with its content reaching a maximum on the 22nd day and then decreasing rapidly. Cycasin content increased gradually after shoot elongation ceased, showing a maximal value of 2 mmol/100g in about 2 months, then decreased gradually along with leaf weight loss. Although azoxyglycosides were present in the middle axes of cycad leaves, only leaflets were analyzed because growing axes became rigid and could not be completely macerated for satisfactory glycoside extraction.