AbstractAn HPLC method suitable for the determination of canavanine and canaline in jack beans (Canavalia ensiformis (L)DC), chick serum and excreta is described. Canavanine and canaline at ‘on column’ levels of 1–300 pmol were readily detected and quantified fluorimetrically as their orthophthaldehyde (OPA)‐2‐mercaptoethanol adducts. Linearity of response was obtained for both amino acids. Good recoveries of supplemental canavanine and canaline (mean recovery = 97.5%) were also obtained for both amino acids. Analysis time for canavanine was about 6 min using an ODS‐Hypersil column (dp = 3 μm; 5 mm id by 100 mm long). Analysis of both amino acids simultaneously using a Spherisorb ODS2 column (dp = 5 μm; 5 mm id by 160 mm long) took about 8 and 40 min respectively. The Spherisorb column did not prove satisfactory for the analysis of canavanine in serum. Canavanine was present in jack beans and the serum of chicks fed jack beans but canaline was not detected in either. Canavanine and canaline were not detected in excreta from chicks fed a j