Background: The pathways of resistance to atazanavir (ATV) and amprenavir (APV) converge at position 50 of HIV protease. The determinants of cross-resistance were analysed during in vitro sequential or concomitant combination pressure with both drugs. Methods: Recombinant viruses containing in vitro and in vivo selected I50L and I50V proteases were constructed and cultured in increasing concentrations of APV or ATV, respectively. In addition, in vitro resistance to ATV plus APV was performed. All the resistant viruses obtained were genotyped and phenotyped. Results: ATV or APV alone selected I50L- or I50V-containing variants. Subsequent addition of ATV to I50V-containing recombinant viruses led to the reversion of this change and the later selection of I50L. By contrast, addition of APV to I50L-containing recombinant viruses was not associated with reversion. The combined pressure with ATV plus APV selected several changes but not at position 50. Phenotypically, both sequential and concomitant ATV-APV pressure yielded viruses resistant to all the drugs tested, although the emergence of I50L by ATV pressure on APV-resistant variants was associated with a reduced resistance to APV and darunavir. Conclusions: All drug combinations led to APV plus ATV cross-resistance. The different pathways select for isoleucine or leucine at position 50, whereas the I50V mutation was excluded. Sequential pressure with ATV might have an advantage in terms of modulating the sensitivity of HIV to other protease inhibitors. ©2010 International Medical Press.