Human colon-carcinoma cells were exposed to D-glucosamine at 2.5, 5 and 10 mM, concentrations that were growth-inhibitory but not cytocidal in the presence of a physiological glucose concentration. Labelling of these HT-29 cells with D-[14C]-glucosamine, followed by nucleotide analyses, demonstrated that UDP-N-acetyl-hexosamines represented the major intracellular nucleotide pool and the predominant metabolite of the amino sugar. D-[14C]Glucosamine was not a precursor of UDP-glucosamine. After 4h exposure to D-glucosamine (2.5 mM), the pool of UDP-N-acetylhexosamines was increased more than 6-fold, whereas UTP and CTP were markedly decreased. UDP-glucuronate content increased by more than 2-fold, whereas purine nucleotide content was little altered. Uridine (0.1 mM) largely reversed the decrease in UTP, CTP, UDP-glucose and UDP-galactose, while intensifying the expansion of the UDP-N-acetylhexosamine pool. Uridine did not reverse the D-glucosamine-induced retardation of growth in culture. A 50% decrease in growth also persisted when uridine and cytidine, cytidine alone, or UDP, were added together with D-glucosamine. The growth-inhibitory effect of the amino sugar could therefore be best correlated with the quantitative change in the pattern of sugar nucleotides, and, in particular, with the many-fold increase in UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine.