The (13)C-isotopomer enrichment of hepatic cytosolic acetyl-CoA of overnight-fed mice whose drinking water was supplemented with [U-(13)C]fructose, and [1-(13)C]glucose and p-amino benzoic acid (PABA) was quantified by (13)C NMR analysis of urinary N-acetyl-PABA. Four mice were given normal chow plus drinking water supplemented with 5% [1-(13)C]glucose, 2.5% [U-(13)C]fructose, and 2.5% fructose (Solution 1) overnight. Four were given chow and water containing 17.5% [1-(13)C]glucose, 8.75% [U-(13)C]fructose and 8.75% fructose (Solution 2). PABA (0.25%) was present in both studies. Urinary N-acetyl-PABA was analyzed by (13)C NMR. In addition to [2-(13)C]- and [1,2-(13)C]acetyl isotopomers from catabolism of [U-(13)C]fructose and [1-(13)C]glucose to acetyl-CoA, [1-(13)C]acetyl was also found indicating pyruvate recycling activity. This precluded precise estimates of [1-(13)C]glucose contribution to acetyl-CoA while that of [U-(13)C]fructose was unaffected. The fructose contribution to acetyl-CoA from Solutions 1 and 2 was 4.0 ± 0.4% and 10.6 ± 0.6%, respectively, indicating that it contributed to a minor fraction of lipogenic acetyl-CoA under these conditions.