We developed an assay system for ornithine aminotransferase (EC 2.6.1.13) using ninhydrin. Pyrroline 5-carboxylate, a product of enzymatic transamination, reacts with ninhydrin under hot acidic conditions to form a reddish pigment soluble in ethanol. The millimolar extinction coefficient of reaction product dissolved in ethanol was 16.5 at 510 nm. Acidification with perchloric acid effectively abolished the interfering color development by L-ornithine and L-glutamate. The paired activity measurement in mouse tissues by ninhydrin and o-aminobenzaldehyde methods showed a good correlation (gamma = 0.985). In our ninhydrin method, stable ninhydrin replaced unstable o-aminobenzaldehyde, and sensitivity was much higher than that with the conventional o-aminobenzaldehyde method.