A sensitive, simple, and instrument-independent method for the visual detection and identification of multiple nucleic acid amplicons by dipstick has been developed. This method is based on nucleic acid hybridization on the dipstick membrane and a signal amplification system to allow visual detection. The importance of nucleic acid testing (NAT) has become increasingly evident during the last decade for many purposes, such as diagnosing viral infections, monitoring antiviral therapy, and improving the safety of blood supplies. NAT combines the advantages of direct and highly sequence-specific detection of the genome of an infectious agent with an analytic sensitivity that is several orders of magnitude greater than that of antigen detection or virus isolation methods. NAT also reduces the risks of viral transmission during the period between infection and seroconversion (34), of infection with immunovariant viruses (20), of immunosilent carriage (6), and of occult carriage (3, 34). Unfortunately, a high investment cost for instruments, a high running cost for reagents, a lack of maintenance support, complex and often labor-intensive procedures that require highly trained personnel, and the need for cold-chain transport and storage of reagents render NAT unaffordable in the settings where it is needed most. These settings include predominantly the resource-limited countries of Africa, Asia, and Latin America with high prevalence of infectious diseases. Consequently, two complementary approaches to lower the costs of NAT have been proposed: pool testing for large-scale screening (26,35) and the development of multiplex assays for the simultaneous detection of several infectious agents (11).Multiplex assays have recently been developed for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1), which are three blood-borne pathogenic viruses of major epidemiological and clinical importance (9,19,21,22). The AMPLINAT MPX assay (21) and the multiplex real-time quantitative reverse transcription (RT)-PCR assay described by Candotti et al. (9) are based on TaqMan fluorescence technology, whereas the transcriptionmediated amplification-based Procleix Ultrio assay (19) relies on chemiluminescence technology for detection. NAT-based assays consist of three basic steps: extraction of nucleic acid, genome amplification mediated by a procedure such as PCR or RT-PCR, and amplicon detection. The first two steps can be performed with relatively inexpensive equipment, including a centrifuge, a water bath, and a thermal cycler. In contrast, the last step requires either time-consuming and labor-intensive electrophoretic separation or expensive equipment to detect fluorescence or chemiluminescence signals. The rapid visual detection of nucleic acid hybridization on a test strip (dipstick) would offer a simple and cost-effective alternative to these highly complex and instrument-dependent commercial methods. Serological testing performed with rapid dipstick tests is instrument independent ...