4-Hydroxybenzoate was activated with coenzyme A by cells of a strictly anaerobic, phenol-degrading mixed culture to 4-hydroxybenzoyl-CoA, which was reductively dehydroxylated to benzoyl-CoA with reduced benzylviologen as an electron donor. The specific activity of the 4-hydroxybenzoyl-CoA ligase in cell-free extracts of the culture was 100-200 nmol min(-1) mg(-1), that of 4-hydroxybenzoyl-CoA reductase 14.5 nmol min(-1) mg(-1). An increased growth yield of the phenol-degrading mixed culture of 1.8 g/mol with 4-hydroxybenzoate in comparison to phenol as the substrate was found previously and indicated energy generation by decarboxylation of 4-hydroxybenzoate. Addition of 4-hydroxybenzoate to cell suspensions of the mixed culture resulted in a rapid increase of the cellular ATP level. The proton ionophore carbonylcyanide m-chlorophenylhydrazone and the H+- ATPase inhibitor dicyclohexylcarbodiimide prevented an increase of cellular ATP levels during 4-hydroxybenzoate decarboxylation, whereas the sodium ionophore monensin and the putative Na+-ATPase inhibitor ouabain revealed no effect. This was taken as good evidence for the generation of a proton gradient across the membrane by decarboxylation of 4-hydroxybenzoate and ATP formation by H+-ATPase.