Reverse transcription is a complicated process that involves at least two cDNA transfer reactions to produce a full-length copy DNA of the retroviral RNA genome. Because one retrovirus particle contains two identical genomic RNA molecules, the transfers can occur in an intramolecular or intermolecular manner. The mechanism of the first transfer step (minus-strand strong-stop cDNA transfer) has been studied previously in detail in transduction experiments with spleen necrosis virus vectors containing genetic markers. Different results have been reported with respect to the type of strand transfer mechanism. In this study, we analyzed the first strand transfer for human immunodeficiency virus type 1 (HIV-1). Two genetically marked genomes were copackaged into virions and reverse transcription was initiated within these particles upon permeabilization by NP-40 and addition of dNTPs. To test whether intrastrand or interstrand transfer had occurred, the cDNA products of this endogenous reverse transcription reaction were extracted from the virions and analyzed for the presence of restriction enzyme recognition sites provided by the genetic markers. The results of this analysis demonstrated that the first DNA transfer reaction occurs in a random manner, with approximately the same contribution of intrastrand and interstrand transfers. The ability to perform intermolecular strand transfer was lost upon extraction of the dimeric RNA template from the virion particle.