Phospholipase C (PLC) activity secreted by bacteria as a virulence factor is commonly detected by use of the artificial substrate p-nitrophenylphosphorylcholine (p-NPPC). We examined several commercially available enzymes (phosphodiesterases, phosphomonoesterases, phospholipase A, lipase, protease) for their hydrolytic activity towards p-NPPC and compared these results with those of PLC tests using phospholipid substrates. Our data indicate that, in addition to PLC, several other enzymes which can affect phosphate eaters are able to hydrolyze p-NPPC. We therefore suggest to use lipid substrates for correct characterization of bacterial PLCs, especially when whole bacteria or crude enzyme preparations are investigated. (C) 2000 Elsevier Science Inc. All rights reserved.