Objective: This study tested the hypothesis that exposure of human molar teeth to tetracycline (TCN) derivatives in vitro results in tooth discoloration resembling the clinical presentation of TCN staining. Method and Materials: The effects of exposure of 20 extracted human molar teeth to distilled water, chlortetracycline, doxycycline, or minocycline were compared. The baseline color of each tooth was analyzed with a dental spectrophotometer. The pulp chambers were each filled with a TCN derivative solution and then sealed. The teeth were placed in a centrifuge tube and then centrifuged at 2,800 rpm for 20 minutes. Color change was monitored weekly for 7 weeks. Digital images of the surfaces were recorded. For each specimen at every evaluation period, color change from baseline was calculated using Commission Internationale d'Eclairage (CIE) Delta E 2000 (∆e00). Results: There was a significant association between the type of derivative used and ∆e00, as well as between the evaluation period and ∆e00. There was also a significant association between the interaction term, derivative 3 evaluation period, and ∆e00. Results of the Holm-Sidak post hoc test demonstrated that all 3 TCN derivatives were associated with significantly larger ∆e00 than the control group (P ¡Ü .05). Conclusions: All 3 TCN derivative solutions produced significant color changes as time progressed. Different TCN derivatives produced a different L* (lightness), C* (chroma), and H* (hue), with minocycline behaving distinctly differently from chlortetracycline and doxycycline. The model could be used to study the underlying mechanisms of TCN staining as well as many aspects of vital tooth bleaching. Keywords: chlortetracycline, doxycycline, minocycline, tetracycline staining, tooth bleaching