A fundamental biological property of retroviruses and RNA viruses is their ability to rapidly mutate and evolve. The ability of these viruses to generate high levels of genetic diversity during replication has clearly had a profound impact on their ability to maintain their niche in nature, and to rapidly adapt to changing environmental conditions or opportunities to expand their host range. Previous reports with HIV-1 have indicated that the cell type in which HIV-1 replicates does not have a profound impact on HIV-1 diversity. However, due to differences in dNTP pool levels and expression levels of HIV-1 DNA editing enzymes, the hypothesis that cell type does influence the diversity of HIV-1 populations was formulated. To test this, a panel of relevant cell types (i.e., CEM-GFP, U373-MAGI, 293T, and SupT1) was analyzed for their ability to influence HIV-1 mutant rate and spectra. No differences were observed in overall mutation rate, but intriguingly, cell type differences impacted HIV-1 mutation spectra. These observations represent the first description of significant differences in HIV-1 mutation spectra observed in different cell types in the absence of changes in the viral mutation rate and, imply that such differences could have a profound impact on HIV-1 pathogenesis, immune evasion, and drug resistance. The most common mutation type that arises during HIV-1 replication is transition mutations, particularly G-to-A mutations. Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) proteins create G-to-A mutations at specific cytosine dinucleotides. In order to better define the locations of APOBEC3G (A3G)-mediated G-to-A mutations, we tested the hypothesis that sequence context and DNA secondary structure influence the creation of A3G-mediated G-to-A mutations. Single-stranded DNA secondary structure as well as the bases directly 3'and 5' of the cytosine dinucleotide were found to be critically important for A3G recognition. These observations provide the first demonstration that A3G cytosine deamination hotspots are defined by both sequence context and the single-stranded DNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on the generation of HIV-1 diversity, ultimately influencing the biological properties of the progeny virus variants.