1. To evaluate the condition under which net glucose production from acetone, added as sole substrate, occurs different pretreatments of mice, in combination with starvation, were used; (i) acetone pretreatment (acetone is a known inducer of cytochrome P-450 isozymes involved in this pathway), (ii) fructose pretreatment (to induce NADPH+H+ generating enzymes) or (iii) their combination. 2. There was net glucose formation from acetone only in that case, when the cells were prepared from 48 hr fasted animals pretreated with both acetone and fructose. However, using 2-14C-acetone, incorporation of 14C-carbon into glucose could be detected in all the cases and, at the same time, acetone was without any effect on protein synthesis. 3. The addition of acetone increased gluconeogenesis from alanine in almost all the cases. The only exception from this general rule was that the case, when hepatocytes were prepared from acetone pretreated 48 hr starved mice where, instead of the elevation of glucose formation, a decrease of that was caused by acetone. 4. Acetone decreased 14C-carbon incorporation into glucose from 14C-(U)-alanine added at saturating concentration in hepatocytes prepared from starved mice. 5. Similarly to acetone there was no net glucose formation from acetone either when added alone, however, it enhanced gluconeogenesis from alanine at non-saturating concentrations of the amino acid. 6. Methylglyoxal proved gluconeogenic in all the cases. 7. It is concluded that net glucose formation from acetone as sole substrate occurs only under those conditions which are far from a physiological situation, however, when gluconeogenesis from another substrate takes place, acetone can contribute to net glucose formation in hepatocytes prepared from fasted mice.