Immune-stimulating complexes (ISCOMs) are stable colloidal complexes of the adjuvant Quil A, cholesterol and phospholipid, which are effective carriers for subunit vaccines. The techniques currently available for the preparation of ISCOMs from the constituent components are rather complex and are based on either centrifugation or dialysis. This note reports a new simple procedure for the preparation of ISCOM matrices based on hydration of a cholesterol/phospholipid film with an aqueous solution of Quil A. It is demonstrated that ISCOM matrices do not form in the absence of phospholipid when prepared by this method. Further, the ratio by weight of phospholipid to either cholesterol or Quil A must be greater than that required for preparation by either dialysis or centrifugation. Photon correlation spectroscopy, negative stain transmission electron microscopy and centrifugation through a sucrose gradient demonstrate that ISCOM matrices can be prepared from cholesterol/lipid films by hydration with an aqueous solution of Quil A when the ratio of phospholipid:cholesterol:Quil A by weight is 6:1:4, respectively. Lower ratios of phospholipid:cholesterol reduce the efficiency of ISCOM formation while higher ratios produce systems containing a mixture of ISCOMs together with liposomes. Copyright (C) 2000 Elsevier Science B.V.