Abstract A new assay for phospholipase C activity that uses alkaline phosphatase to convert phosphorylcholine to inorganic phosphate is described. The determination of inorganic phosphate is performed in the presence of phosphatidylcholine and protein after the addition of sodium dodecyl sulfate. Phospholipase C activity determined by this coupled enzyme assay agrees well with data obtained by extracting and measuring phosphoryl[ 14 C]choline produced from phosphatidyl[ methyl - 14 C]choline. The assay is sensitive to 1 nmol of phosphate, requires no removal of protein or phospholipid, and will work with a variety of phospholipid substrates. The assay is faster and more sensitive than previously published procedures. Stimulation of phospholipase C from Clostridium perfringens by ammonium sulfate is also reported.