Lentiviral vectors derived from human immunodeficiency virus (HIV) have demonstrated exceptional promise as tools for gene therapy applications (1,2), but have also raised safety concerns because of potential creation of replication-competent lentivirus (RCL) by uncontrolled recombination (3–6). In order to be safe for clinical use, vector preparations must be formally and extensively tested to show they are absolutely free of RCL. Various methods have been proposed to detect RCL, all of which report high specificity and sensitivity and all of which can discriminate between replication-defective virus and RCL. There is currently a compelling need for standardization and validation of this method.