d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [(14)C]Uridine labelling of hepatoma cells indicated a division of [(14)C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmolxh(-1)x(kg AS-30D or liver wet wt.)(-1). As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of (14)CO(2) into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5'-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37 degrees C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.