In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine-xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine-xylazine. Optical imaging using a charge-coupled-device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest.