Five-year survival for non-small cell lung cancer is 15%. Gemcitabine is a nucleoside analogue that inhibits ribonucleotide reductase and interferes with DNA replication. In this study, we sought to compare short versus continuous infusion gemcitabine in an in vitro bioreactor system using pharmacokinetic-guided dosing. Gemcitabine was infused over either 0.5 or 2.5h to produce concentration-time profiles that mimic those measured in biological samples (i.e., patient plasma). The effects of gemcitabine on the growth and survival of H2009 cells were examined using trypan blue staining, cell cycle analysis, TUNEL assay, and clonogenic assay. Data were analyzed with two ways analysis of variance. Maximum gemcitabine (Cmax) concentrations during the short infusion were 51.2+/-10.4 microM and for the continuous, 14.8+/-2.93 microM. Steady-state concentrations during the continuous infusions were 14.9+/-2.90 microM. Gemcitabine treatment resulted in a decrease for G1 fraction relative to controls. G2/M, subG1 and TUNEL were higher following gemcitabine relative to controls. Survival was approximately 20-fold higher following the short infusion compared with the continuous infusion (p = 0.0085). In conclusion, gemcitabine infused by this novel method induced apoptosis after both the short and continuous infusions, and long-term survival was significantly diminished following continuous compared with the short infusion.