It has been proposed that key enzymes of ureogenesis and the alanine aminotransferase activity predominate in periportal hepatocytes. However, ureogenesis from alanine, when measured in the perfused liver, did not show periportal predominance and even the release of the direct products of alanine transformation, lactate and pyruvate, was higher in perivenous cells. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea productions from precursors such as lactate or pyruvate plus ammonia. In the present work these aspects were investigated using the bivascularly perfused rat liver. The results of this study confirm that gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region. Alanine synthesis from lactate and pyruvate + ammonia, however, predominated in the perivenous region. Furthermore, no predominance of ureogenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. These observations corroborate the view that data on enzyme activity or expression cannot be extrapolated unconditionally to the living cell. The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureogenesis in periportal cells. However, since urea synthesis occurs at high rates in all hepatocytes with the possible exclusion of those cells not possessing carbamoyl-phosphate synthase, it is probable that ureogenesis is equally important as an ammonia-detoxifying mechanism in the perivenous region.