A heterogeneous series of compounds, derived from p-aminobenzoic acid (PABA), has been investigated for their PABA-antagonistic potency in a cell-free H2-pteroate synthesizing system of E. coli. A prerequisite of compounds, other than sulfones or sulfonamides, to compete with PABA for the enzyme H2-pteroate synthetase appeared to be the presence of a p-aminobenzoyl moiety. Substitution of the carboxyl group of PABA by an ester, an amide, or a ketone function, however, strongly reduces the ability to interact with the PABA binding site on the enzyme. This decrease in affinity probably has to be ascribed to the inability to create a sufficient negative charge in the carbonyl part of these p-aminobenzoyl derivatives. The relatively high affinities of L-PABG (16), PABP (22), and the alpha-phenyl derivative of 22, as compared with the other substituted p-aminobenzamides and p-aminobenzene-1-alkanones, are explained by assuming that these compounds, besides interfering with the PABA receptor site, also interact with an accessory area on the enzyme.