Rate constants for Cs-proR/proS-hydron exchange from L-dihydroorotate were determined by H-1 NMR and detritiation in various oxygen-containing and amine buffers at 37 degrees C and ionic strength 1.0 M in aqueous solution. Thermodynamically unfavorable proton transfer from the C-5-proR and -proS positions (pK(a) = 20-21) to oxygen-containing and amine bases shows general-base catalysis with a Bronsted beta value of 0.84 +/- 0.05 and 0.81 +/- 0.08, respectively, which is consistent with a late, enolate-like transition slate. General-base catalysis is detectable because there is a 160- or 85-fold negative deviation for the C-5-proR and -proS protons, respectively, from this correlation for deuterioxide ion. Deviations of (k(H)/k(T))(obsd) and (k(D)/k(T))(obsd) from the Swain-Schaad equation are consistent with internal return of the transferred hydron to a free C-5-carbanion/enolate intermediate from the conjugate general acid. This corresponds to an Eigen-type mechanism for hydron transfer, in which both proton transfer and diffusional separation of the C-5-carbanion/enolate conjugate general-acid complex are partially rate-limiting, and a modest intrinsic barrier for C-5-hydron exchange. it is concluded that the C-5-carbanion/enolate can have a significant lifetime in aqueous solution and on dihydroorotate dehydrogenases (EC 1.3.1.14; ECT 1.3.99.11).