The orotate pathway is generally accepted as the major source of the pyrimidine components of DNA and RNA in mammalian tissues. However, it has not been established whether extrahepatic tissues utilize this pathway or depend upon the liver for their pyrimidine requirements. The exclusive source of the carbamoylphosphate incorporated into pyrimidines is currently assumed to be the glutamine-dependent carbamoylphosphate synthetase (CPS-II), an assumption based upon the fact that CPS-II is localized with the other enzymes of the orotate pathway in the cytosol. The more active, ammonia-dependent, acetylglutamate- activated enzyme (CPS-I) is localized with ornithine carbamoyltransferase in the mitochondrial fraction of the liver and, therefore, assumed to be the exclusive source of carbamoylphosphate utilized in the biosynthesis of urea. Since the activity of CPS-I greatly exceeds that of CPS-II, and since the occurrence of the complete orotate pathway in extrahepatic tissues remains to be established, we propose an investigation with the following specific aims: (a) An evaluation of the factors controlling the availability of intramitochondrial carbamoylphosphate for extramitochondrial reactions. (b) A study of the participation of CPS- I in hepatic pyrimidine biosynthesis. (c) An investigation of the role of the liver in providing pyrimidines or their precursors for nucleic acid synthesis in extrahepatic tissues. (d) A study of the occurrence of the orotate pathway in extrahepatic tissues. (e) Studies on the regulation of pyrimidine biosynthesis. These studies will provide a clearer understanding of the control of pyrimidine biosynthesis in mammals and role of liver in the growth and development of extrahepatic tissues.