Several tests are currently available to assist in the diagnosis of the hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Tests that actually detect or quantify these viruses are based on the polymerase chain reaction (PCR) technique. However, the application of PCR is limited by the cost, labor, time-consumption, and potential for contamination. In this article we describe some procedures developed to reduce these limitations. We have developed and validated simultaneous detection methods for HIV RNA and HCV RNA in single serum samples using Amplicor PCR tests. The sensitivity and specificity of this method are comparable with the results obtained with commercial reverse transcription polymerase chain reaction (RT-PCR) techniques for HIV and HCV RNA detection. In addition we have modified the HIV Amplicor test for the RT-PCR procedure and the Chomczynski's method of RNA isolation. We hope that our method can find same applications in HIV and HCV coinfection research, blood screening, and medical diagnosis.