The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecular-weight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription.