The measurement of argininosuccinate lyase (ASase) and arginase, both in liver and erythrocytes, was developed by using a commercial amino acid analyzer. The method is based upon the use of two different substrates, argininosuccinate and arginine for ASase and arginase, respectively, and the measurement of only one final metabolite: ornithine. The use of ornithine as a marker of biological activity of ASase is related to the fact that in the urea cycle, the specific activity of arginase is much higher than that of ASase; thus, during in vitro determinations, arginine, which is the product of ASase, is rapidly converted to ornithine. The sensitivity of the methods is very high since we were able to detect both activities using very diluted rat liver homogenates (0.10 mg protein/ml) or few microliters of human blood. In rat liver the Vmax for ASase and arginase were respectively 0.54 and 140 mumol/h/mg protein; the apparent Km values 1.25 and 13.5 mM. In human erythrocytes the Vmax for the same enzymes were 7.2 and 170 nmol/h/mg Hb and the apparent Km values were 0.66 and 9.5 mM. In 10 healthy volunteers the specific activity of ASase and arginase determined in blood were respectively 8.60 +/- 0.46 and 124.1 +/- 14.5 nmol/h/mg Hb. The results obtained from 2 patients suffering from argininosuccinic aciduria were also reported. In these latter cases while ASase was not detectable in blood, arginase activity was at the lowest end of the confidence limits determined in healthy volunteers.