The agonist-induced breakdown of phosphoniositides by a specific phosphatidylinositol phosphodiesterase (phosphatidylinositol-specific phospholipase C) has been suggested to be a receptor-coupled phenomenon and a critical step for the initiation of subsequent cellular processes (Michell, 1982; Hasegawa-Sasaki & Sasaki, 1982; Berridge, 1984). In the majority of cases this enzyme has been located in the soluble fraction of the cell and when assayed also appears to hydrolyse the polyphosphoinositides (Low & Weglicki, 1983; Wilson et al., 1984). We confirm here the presence of a phosphatidylinositol-specific phospholipase C isolated from the soluble fraction of pig mesenteric lymph-node lymphocytes, as initially reported by Allan & Michell (1974), and subsequently describe the steps taken to produce a partially purified phospholipase C, revealing multiple forms of the enzyme. Phospholipase C activity was isolated from lymphocytes by the method of Allan & Michell (1974). Ninety-six per cent of the activity was found in the soluble fraction; 4% could be detected associated with the membrane pellet when homogenized with 1.0% (v/v) Triton X-100. The soluble enzyme was found to be specific for phosphatidylinositol when assayed with a range of phospholipids. Before purification, the crude phospholipase C preparation was studied to find the optimal assay conditions for this enzyme. Phospholipase C activity was measured by its ability to hydrolyse a mixture of phosphatidyl [2-3H] inositol (Amersham International, Amersham, Bucks., U.K.), approx. 15000 d.p.m., and 150nmol of phosphatidylinositol (Lipid Products, Redhill, Surrey. U.K.,) resuspended in 0.5 ml of 50 mM-Tris/malate/NaOH buffer, pH 5.5, containing 1 mM-CaC12. Incubations were carried out at 37°C for 1Omin and stopped by extraction with 2 ml of chloroform/methanol/HCI ( 1 00 : 200 : 1, by vol.) followed by 0.6 ml of chloroform and 0.61111 of 0.05 M NaCl. One millilitre of the aqueous phase was removed for scintillation counting to determine the radioactivity released. Phospholipase C activity could be detected in the absence of any additional divalent cations, presumably due to their presence within the crude enzyme preparation as 1 mM-EDTA virtually abolished all the enzyme activity. Under the standard assay conditions, presence of 1 mMCaClz gave optimal activity. The pH optima in the absence of additional CaClz were found at pH 5.5 and pH 7.0, confirming the report of Allan & Michell (1 974). With the addition of 1 mM-CaC12 the activity was increased considerably and the two distinct pH optima were replaced by a broad peak at pH 6.0, resulting in an overall 2-fold increase in the activity at pH 5.5 and a 4-fold increase in the activity at pH 7.0. The initial step in the purification procedure employed gel filtration on a column (90cm x 2.5 cm) of Sephadex G200 (Pharmacia Fine Chemicals, Milton Keynes, Bucks., U.K.). The column was eluted with 5OmM-Tris/HCl, pH 7.2/0.1 mM-PMSF/O.I mM-DTE at a flow rate of 10ml/h to give the elution profile seen in Fig. 1. Four peaks of