Dietary folates exist largely as polyglutamates that require deconjugation prior to absorption. This process is catalyzed by intestinal pteroylpolyglutamate hydrolase (PPH) mainly associated with the jejunal brush border membrane. Previous studies have shown that citrate ion as well as the soluble fraction of many foods can inhibit PPH in vitro. This study was conducted to characterize further the in vitro inhibition of porcine PPH by fractions of selected foods (orange juice, tomatoes, and lima beans) and by organic anions to evaluate potential effects on bioavailability. Organic anions tested were competitive inhibitors with respect to the polyglutamyl folate substrate, with the following K(i) values: citrate, 6.42 mmol/L; malate, 10.1 mmol/L; phytate, 6.48 mmol/L; ascorbate, 19.6 mmol/L. Neutralized orange juice in the reaction mixture strongly inhibited PPH activity, while neutralized tomato homogenate caused weaker inhibition and lima bean homogenate inhibited much more weakly, all with kinetics indicating competitive inhibition. Fractionation of food extracts indicated that the inhibitors were anions of low molecular mass (<6-8 kDa). Chromatographic separation followed by in vitro assay indicated that citrate was the major inhibitor, with lesser inhibition by malate and phytate. These results indicate the potential for dietary constituents to retard the action of intestinal brush border PPH, with possible inhibitory effects on the bioavailability of polyglutamyl folates. The in vivo significance of this effect would depend on the extent of inhibition relative to the total intestinal PPH activity.