The reverse transcriptase (RT) of HIV-1 has a non-templated addition (NTA) activity and can perform template switches with a very short (even two nucleotides) complementarity between the 3'-ends of the primer donor strand and the template acceptor strands [1]. We have studied how the combination of several pivotal parameters can all lead to strand switches during DNA synthesis by HIV-1 RT. These include dNTP bias in the NTA step, the availability of acceptor strands with 3'-end sequences complementary to the de novo-generated primer tails and the stabilities of the clamped duplexes formed between these primer tails and the acceptor strands. (C) 2011 Elsevier Inc. All rights reserved.