The inhibitory effects of nucleotides and related substances on rat hepatic UDP-glucuronosyltransferase (UGT) were studied. ATP and NADP+ markedly reduced 4-methylumbelliferone (4-MU) UGT activity only when detergent-treated rat liver microsomes were used as the enzyme source. The IC50 values of adenine, ATP, NAD+ and NADP+ were estimated to be below 20 microM, whereas AMP had no inhibitory effect. From the kinetic behavior observed, these adenine-related compounds were assumed to inhibit UGT activity non-competitively without competing with either 4-MU or UDP-glucuronic acid. Among guanine, cytosine and their related nucleotides, only triphosphate nucleotides (CTP and GTP) exhibited potent UGT inhibition, although the effect of GTP was weak. Estradiol 3- and 17-glucuronidation were also inhibited by the inhibitors of 4-MU UGT. The only exception was that estradiol 17-glucuronidation activity was inhibited by AMP (IC50=31 microM). In addition, AMP antagonized the inhibitory effects of adenine, ATP, and NADP+ on 4-MU and estradiol 3- glucuronidation activities. These results suggest that (1) a number of cellular nucleotides present within the endoplasmic reticulum regulate UGT function; and (2) these substances bind to a common allosteric site on UGT to reduce catalytic function.