The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg²⁺ or Mn²⁺. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new ³²P-based, Mg²⁺-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID₅₀s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID₅₀ for simian immunodeficiency virus isolated from a rhesus macaque (SIV_mac). Analysis of recombinant HIV-1 RT enzyme indicated that 10⁵ U, which is equivalent to 4.25 × 10⁴ virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg²⁺-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans.