Expression of the firefly luciferase gene in transgenic plants produces light emission patterns when the plants are supplied with luciferin. We explored whether in in vivo pattern of light emission truly reveals the pattern of luciferase gene expression or whether it reflects other parameters such as the availability of the substrate, luciferin, or the tissue-specific distribution of organelles in which luciferase was localized. The tissue-specific distribution of luciferase activity and the in vivo pattern of light were examined when the luciferase gene was driven by different promoters and when luciferase was was redirected from the peroxisome, where it is normally targeted, to the chloroplast compartment. It was found that the distribution of luciferase activity closely correlated with the tissue-specific pattern of luciferase mRNA. However, the in vivo light pattern appeared to reflect not only tissue-specific distribution of luciferase activity, but also the pattern of luciferin uptake.