Three novel 4-subsituted-7-(2′-deoxy-2′-fluoro-4′-azido-β-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine analogues were designed, synthesized, and tested for their anti-HIV-1 activity. Initial biological studies indicated that among these pyrrolo[2,3-d]pyrimidine ribonucleoside analogues, 4-amino-7-(2′-deoxy-2′-fluoro-4′-azido-β-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine 10 exhibited the most potent anti-HIV-1 activity (EC 50 =0.5±0.3μM), while 4-hydroxy-7-(2′-deoxy-2′-fluoro-4′-azido-β-d-ribofuranosyl)pyrrolo[2,3-d] pyrimidine 9 and 4-amino-5-fluoro-7-(2′-deoxy-2′-fluoro-4′-azido-β-d-ribofuranosyl)pyrrolo[2,3-d] pyrimidine 11 showed moderate activity (EC 50 =13±8 and 5.4±0.3μM, respectively). The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds with concentrations up to 25μM.