Human immunodeficiency virus (HIV) is a cytoplasmic retrovirus which is transmitted via body fluids, especially through blood products and sexual contact, and is the causative agent of the acquired immunodeficiency syndrome (AIDS). Only about 5 to 10% of the patients infected with HIV contract AIDS; the great majority of infected people either develop a less aggressive condition (AIDS-related complex) or appear healthy. All persons infected with HIV may transmit the virus. In order to protect the national blood supply and to help in diagnosis, tests have been developed to identify infected persons. These include viral isolation techniques, enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), radioimmune precipitation (RIP) assay, Western blot, and, most recently, antigen identification and gene probes. Although the sensitivity and specificity among these methods varies, all are susceptible to false-positive and/or false-negative results. In order to understand the reasons for fragility in methodologies, it is necessary to appreciate several basic concepts related to the biochemistry, biology, pathophysiology, and genetic characteristics of HIV and related viruses. The purpose of this review is to present the strengths and weaknesses of each method, with emphasis on peculiar viral characteristics that lead to methodological defects or efficacies.