Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus-based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfer nearly always occurred precisely at the 5' end of genomic RNA. This finding allowed us to use first strand transfer to study the classes of errors that reverse transcriptase can and/or does make when it switches templates at a defined position during viral DNA synthesis. We found that errors occurred at the site of first strand transfer approximately 1000-fold more frequently than reported average reverse transcriptase error rates for template-internal positions. We then analyzed replication products of specialized vectors that were designed to test possible origins for the switch-associated errors. Our results suggest that at least some errors arose via non-templated nucleotide addition followed by mismatch extension at the point of strand transfer. We discuss the significance of our findings as they relate to the possible contribution that template switch-associated errors may make to retroviral mutation rates.