Viral production and variability of HIV-1 is normally high in vivo causing the necessary conditions for cellular superinfection. In order to evaluate the superinfection dynamics in vitro, H9HTLVIIIB cell line was superinfected with HIVMN. Superinfected cells showed nearly 50% cell mortality at day 1 post-superinfection (ps), which increased significantly up to day 4 ps. Superinfecting genome was detectable until day 10 ps. The superinfecting strain was found in the supernatant only on day 1 ps, but was recovered up to day 4 ps by coculture with non-infected cells. The existing strain (HIVHXB2) was recovered throughout the studied period. Pseudotype formation by the HIVHXB2 genome and envelope proteins of the superinfecting strain (HIVMN) was observed from day 1 to 6 ps. Viral production was increased by 1.7 LOG in superinfected cells from day 1 ps. Both viral production increase and pseudotype formation could be relevant for HIV pathogenesis in vivo.