Introduction: Various techniques are available for in vivo imaging, and precise understanding of their characteristics is essential for effective use of the imaging results. We established human mesothelioma cell lines expressing red fluorescent protein (RFP) and examined their fluorescence intensity and uptake of positron emission tomography (PET) tracer analogs to compare their characteristics and assess their usefulness in the evaluation of therapeutics. Method: A human mesothelioma cell line was stably transfected to express RFP. Fluorescence, cell number and protein amount were measured during cell growth and treatment with cytotoxic reagents. In in vivo experiments, RFP-expressing cells were injected subcutaneously or into the pleural cavity of nude mice, and fluorescence images were taken with or without pemetrexed treatment. The uptake of [3H]3′-deoxy-3′-fluorothymidine ([3H]FLT) and [14C]2-fluoro-2-deoxy-d-glucose ([14C]FDG) under treatment with the above reagents in vitro and in vivo were examined. Results: Strong correlation was observed between fluorescence intensity and total cell number with or without cytotoxic treatment. The uptake of [3H]FLT and [14C]FDG decreased rapidly after the initiation of treatment with actinomycin D or cycloheximide. When treated with pemetrexed, the uptake of [3H]FLT temporarily increased. The cells formed subcutaneous and orthotopic tumors, with fluorescence intensity correlating with tumor volume. The correlation was sustained under pemetrexed treatment. The uptake of [3H]FLT in vivo increased significantly early after pemetrexed treatment. Conclusion: Fluorescence imaging could be used to semiquantitatively monitor tumor size, whereas PET could be used to monitor tumor response to therapeutic treatments, and especially, FLT might be a good marker of the response to anti-folate chemotherapeutics. © 2008 Elsevier Inc. All rights reserved.