The agonistic and antagonistic effects of nuclearly substituted p-aminobenzoic acids (PABA) on the folate-synthesizing system of E. coli have been studied in whole cell and cell-free systems. All studied derivatives form dihydropteroic acid analogues in the presence of a cell-free folate-synthesizing enzyme system. A thin-layer chromatographic system has been elaborated to determine the rate of analogue formation in the cell-free system. Physicochemical parameters of the PABA derivatives, such as pKa, pi, and Rm values, have been determined. These values have been used in a structure-activity analysis which revealed that the rate of analogue formation in the absence of PABA is independent of the lipophilic properties. Ionization seems to be the decisive factor for the incorporation. As all studied PABA derivatives are totally ionized under the experimental conditions, the rates of analogue formation are very similar with the exception of compounds bearing bulky groups in the 2 position. The variance in inhibitory power may therefore either be due to differences in the ability of the analogues to serve as metabolites or to competition with PABA.