Cleavage of the carbon-phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l-1 and 100 mmol l-1, respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity for this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.