Human immunodeficiency virus type 1 (HIV-1) genetic diversity is one of the most important features of HIV-1 infections and the result of error accumulation during reverse transcription and of high viral turnover. HIV-1 reverse transcription is influenced by factors such as the level of nucleotides and/or the cellular activation state. HIV-1 diversity was investigated after 48 h of viral propagation in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors in three different cell culture conditions: (1) resting PBMCs, (2) simultaneous infection and PBMC activation, and (3) PBMC activation 72 h before infection. Cellular DNA was extracted and proviruses of each culture condition were amplified. Single-genome PCR clones were obtained and the protease and reverse transcriptase of the pol gene were sequenced. An elevated number of nucleotide substitutions in all three culture conditions were observed. In condition 1, the mutational rate observed ranged from 1.0 × 10(-3) to 2.1 × 10(-2), the genetic diversity was 0.6%, and hypermutation was observed in 7.1% of sequenced clones. In condition 2, the mutational rate ranged from 1.0 × 10(-3) to 1.0 × 10(-2), the genetic diversity was 0.8%, and hypermutation affected 6.7% of clones. In condition 3, the mutational rate ranged from 2.8 × 10(-3) to 1.1 × 10(-2), the genetic diversity was 1%, and 5.9% of clones were hypermutated. Substitutions occurred more frequently in some specific nucleotide stretches, and a common pattern for substitutions in all the different conditions was identified. There was a significant accumulation of mutations during the initial periods of in vitro HIV-1 propagation irrespective of culture conditions. The rapid accumulation of virus diversity might represent a viral strategy when colonizing new hosts. Complementary studies are necessary to allow for a better understanding of the initial periods of infection, which represent a crucial event related to disease progression.