Since animal testing should be avoided whenever possible, the development of in vitro tests for predicting the effect of chemicals becomes a major field. This rise of in vitro test systems led to an increased requirement for well-characterized continuously growing cell lines. Monitoring of the cells during test and routine culture is necessary to gain relevant and reproducible results. In the present study, the influence of passaging under constant culture conditions on the human keratinocyte cell line HaCaT was investigated. Data demonstrated that growth rate rose with increasing passages. Doubling times of the cells were decreased to 24 ± 0.6 h in the late passages (12-16), in comparison to 36.2 ± 1.5 h in the early passages (2-8). These data were confirmed by a fall in mRNA expression levels of keratin 1 and transglutaminase 1 within the passages. Furthermore, the activities of the xenobiotic metabolizing phase II enzyme N-acetyltransferase 1 (NAT1) were higher in the late passages compared to the early passages. These results are contrary to an expected decrease in enzyme activity and proliferation rate induced by replicative senescence or cell aging. Data also indicate that routine culture might result in significant changes in proliferation and phase II metabolism. These findings reinforce the necessity of a strict characterization and knowledge of regulation of in vitro systems, as well as the need for new biomarkers, in order to use cells for the development and evaluation of reproducible in vitro test systems.