The most direct test of functional capacity of the liver in nitrogen disposal is to stress the urea cycle with a high protein load. This has been used in the diagnosis of heterozygosity for ornithine carbamoyltransferase deficiency for many years by measuring the subsequent excretion of orotic acid in urine. Reports have shown some ambiguity in both this and the more recent allopurinol test. We investigated the effects of different foods as the protein load and of different analytical methods. A standardized protocol was developed, giving 35 g protein per m2 surface area as steamed fat-free chicken breast to be eaten within 30 min. Urine was collected at zero time and over 0-2, 2-4 and 4-6 h. Compliance was checked by assessing excretion of amino acids. Diagnostic sensitivity was improved by reference to the change in excretion, i.e. the ratio of excretions 2-4 h/0-2 h. Extension of the test to 6 h gave no diagnostic advantage over a 4 h test. Comparison of the analysis of total orotic acids by the photometric method of Harris and Oberholtzer, the reference method for this study, with that by the method of Goldstein and colleagues showed that the latter gave erratic results with some false positives. However, comparison of the method of Harris and Oberholtzer with specific orotic acid analysis by a modification of the stable-isotope internal standard method of Rimoldi and colleagues yielded the same diagnoses. The improved protein load test gave a clearly positive result in all 16 obligate heterozygotes and 2 possible heterozygotes tested from 14 kindred, and a clearly negative result in all 18 control subjects and all 6 of the possible heterozygotes who were later shown by DNA studies not to carry the family mutation. The test appears at least as sensitive and specific as the allopurinol test, and is more convenient because of the short period of sample collection.