Our aim was to identify whether zidovudine has a role in the emergence of the K103N resistance mutation in the HIV-1 reverse transcriptase gene on non-nucleoside reverse transcriptase inhibitors (NNRTIs). No difference was found in the exposure to zidovudine or major zidovudine mutations between the resistance patterns K103N-/Y181C+, K103N+/Y181C- and K103N+/ Y181C+, either in group A (patients on nevirapine and previously NNRTI naive) or in group B (on any NNRTI and experience of two or more NNRTIs including nevirapine). Group B patients had the highest prevalence of K103N+/Y181C+. In conclusion, zidovudine seems not to determine the emergence of K103N; however, there appears to be an accumulation of NNRTI resistance mutations with sequential use of NNRTIs.