1- or 9-(2-Deoxy-2,2-difluoro-β-L-ribofuranosyl)-thymine, -cytosine and -adenine were synthesized and their in vitro activity against HIV were evaluated. L-Nucleosides have been reported to show interesting anti-HIV and anti-HBV activities, with (-)-(2R,5S)-1-[(2-Hydroxymethyl)oxathiolan-5-yl]cytosine (3TC), (-)-β-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), 5-fluoro-2',3'-dideoxy-β-L-cytidine (L-FddC), and 2'-fluoro-5-methyl-β-L-arabinofuranosyluracil (L-FMAU) being the most promising ones undergoing preclinical and clinical evaluations. In view of the biological activities of 2',2'-difluoro-substituted D-configuration nucleosides and the potent activities of L-nucleosides, this communication reports the synthesis of the corresponding L-isomers (2'-deoxy-2',2'-difluoro-β-L-ribofuranosyl nucleosides) and their preliminary in vitro anti-HIV activities in acutely infected peripheral blood mononuclear (PBM) cells. The synthesis involved preparing (S)-2,3-O-Isopropylideneglyceraldehyde from L-gulonic-γ-lactone, coupling via Reformatzkii conditions to get diastereomers, separating them, and conducting subsequent reactions (hydrolysis, ring closure, silylation, reduction, mesylation, condensation with silylated bases) to obtain nucleosides. Purine derivatives were synthesized using Mitsunobu conditions. Anomeric configuration was assigned based on H-1' splitting pattern. Anti-HIV activities were evaluated in human PBM cells. Among the compounds, adenosine derivative 21 exhibited moderately potent anti-HIV activity without cytotoxicity up to 100 μM in PBM and Vero cells. This encouraging activity prompts study of comprehensive structure-activity relationships for this class of compounds, which is in progress.