Zidovudine and other nucleoside analogue reverse transcriptase inhibitors (NRTIs), like zalcitabine and didanosine used for treatment of individuals infected with HIV-1, can select for viruses with Q151M and other associated mutations (for example, A62V, S68G, V751, F77L, F116Y) in the reverse transcriptase (RT) enzyme. These mutations confer resistance to multiple nucleoside analogues, and thereby compromise the efficacy of this class of drugs. Presently available phenotypic assays for detection of multiple nucleoside analogue resistant (MNR) HIV-1 require testing for each NRTI individually. Here we report an enzymatic RT assay that uses resistance to zidovudine triphosphate (zidovudine-TP) as a diagnostic biochemical marker of MNR HIV-1. This assay exploits the different biochemical mechanisms for zidovudine-resistance conferred by either Q151 M or T215Y/F mutations and the inability of conventional RT assays to detect T215Y/F-associated zidovudine resistance. The assay detects RT activity directly in plasma by using Amp-RT, an ultra-sensitive PCR-based RT assay. We show that enzymatic resistance to zidovudine-TP is specific to MNR RT and is distinguishable from both wild-type (WT) and RT containing classical zidovudine-resistant mutations (D67N, K70R, T215Y/F, K219Q). Compared to WT, MNR HIV-1 RT had 5- to 36-fold increases in the concentration of drug required to inhibit 50% (IC50) of RT activity, depending on the presence of Q151 M alone or with additional MNR mutations. A screening assay utilizing 1 microM zidovudine-TP was developed and validated on 14 reference isolates, 37 plasma specimens, and seven patient-derived viruses. Twenty-three specimens were found to have reduced susceptibility to zidovudine-TP, and all had Q151 M. In contrast, 21 specimens were sensitive to zidovudine-TP, of which 12 had WT genotypes, four had T215Y/F, and five had T69S-insertions along with T215Y/F mutations. This RT-based phenotypic assay provides a specific and rapid tool for the direct identification and monitoring of Q151M-associated MNR HIV-1 in plasma.