We report a multinucleoside resistant (MNR) HIV-1 strain that was susceptible towards tenofovir but acquired phenotypic resistance towards tenofovir during in vitro selective pressure both in the presence and in the absence of tenofovir. The original isolate carried the MNR mutations S68G, V75I, F77L, F116Y and Q151M, the lamivudine mutation M184V, the NNRTI mutation K103N and the yet unreported K70S. After in vitro culturing in the absence of tenofovir, the virus acquired phenotypic resistance towards tenofovir, associated with the acquisition of K70T and the loss of M184V. These changes resulted in higher resistance levels towards zalcitabine, didanosine and tenofovir but lower levels towards zidovudine, stavudine and lamivudine. In vitro culturing in the presence of tenofovir resulted in further enhancement of phenotypic resistance levels towards tenofovir and nucleoside reverse transcriptase inhibitors due to the development of K65R.