The 3'-fluoromodified nucleotide analogs 3'-fluorothymidine triphosphate (FdTTP), 2',3'-dideoxy-3'-fluoro-5-chlorouridine triphosphate (F-5CldUTP), 2',3'-dideoxy-3'-fluoro-5-ethyluridine triphosphate (F-5EtdUTP), 2',3'-dideoxy-3'-fluorouridine triphosphate (FdUTP), and 2',3'-dideoxy-3'-fluoro-5-fluorouridine triphosphate (F-5FdUTP) as well as 2',3'-dideoxythymidine triphosphate (ddTTP), 2',3'-didehydro-2',3'-dideoxythymidine triphosphate (ddeTTP), 3'-chlorothymidine triphosphate (CldTTP), and 3'-rhodanothymidine triphosphate (SCNdTTP) were tested for their ability to inhibit hepatitis B virus (HBV)-associated DNA polymerase activity in vitro. The ID50 values of the most potent inhibitors were 0.15 microM for FdTTP, 0.2 microM for ddeTTP, 0.45 microM for ddTTP, and 0.8 microM for F-5CldUTP. SCNdTTP, CldTTP, and F-5EtdUTP were less efficient (ID50 = 3-5 microM), and FdUTP and F-5FdUTP were the least efficient inhibitors (ID50 = 25 microM) of the enzyme activity. Kinetic analysis revealed a competitive type of inhibition for FdTTP and ddeTTP. The Ki values were estimated to be 0.04 microM and 0.08 microM, respectively, compared with a Km value for dTTP of about 0.18 microM.