Ketonemic states complicating late pregnancy are accompanied by lower brain weights in the newborn. Potential mechanisms whereby ketone bodies might inhibit cell proliferation were therefore examined in the fetal rat brain slice by measuring their impact on the de novo pathway for pyrimidine biosynthesis. DL-beta-hydroxybutyrate (10.8 mM) and acetoacetate (5.4 mM) were both found to diminish the incorporation of NaH14CO3 into [14C]UMP by 30%. This effect was similar in fetal tissues from fed and 48-h starved mothers. Graded concentrations of DL-beta-hydroxybutyrate (1.4-43.2 mM) resulted in a progressive inhibition that could not be explained either by isotope dilution consequent to ketone body oxidation or by a generalized inhibition of protein synthesis. The inhibition was not reversed with 10 mM glutamine, the principal nitrogen substrate for de novo biosynthesis of pyrimidines. When the conversion of orotic acid into UMP was blocked with 6-azauridine, DL-beta-hydroxybutyrate (10.8 mM) inhibited the incorporation of NaH14CO3 into orotic acid by 28%. By contrast, maximally inhibitory concentrations of this ketone body (43.2 mM) had no effect on the incorporation of [6-14C]orotic acid into [14C]UMP. Is is concluded that ketone bodies inhibit the de novo biosynthesis of pyrimidines in fetal brain slices and that they do so at a site proximal to orotic acid formation.