To better understand intracellular and extracellular trafficking of Mycobacterium bovis bacillus Calmette-Guérin (BCG) when used as an intravesical agent in the treatment of transitional cell carcinoma (TCC) of the bladder, recombinant BCG (rBCG) expressing the jellyfish green fluorescent protein (GFP) was created. When the MB49.1 murine TCC cell line was incubated with GFP-expressing rBCG, internalization of the pathogen could be directly visualized by UV microscopy and quantitated by flow cytometry. The in vitro internalization of the GFP rBCG by the bladder tumor cells was temperature dependent, occurring most readily at 37 degrees C and being severely inhibited at 4 degrees C. Optimum internalization was achieved in vitro at a 10:1 BCG-to-tumor cell ratio over 24 h during which approximately 16% of the tumor cells became infected. Cytochalasin B, a phagocytosis inhibitor, abrogated the ingestion by almost 100% at a concentration of 200 micrograms/ml, indicating that contractile microfilaments likely played an important role in this process. By using mitomycin, a DNA cross-linking reagent, to inhibit proliferation of MB49.1 cells, clearance of about 40% of the green rBCG was achieved by 3 days postinfection. No significant difference between the GFP rBCG and wild-type BCG was observed in the ability to induce the expression of cell membrane proteins of major histocompatibility classes I and II, ICAM-I and -II, B7-1 and -2, of Fas from MB49.1 cells or cytokine production from mouse spleen cells. These results indicate that GFP rBCG may serve as a useful substitute for wild-type BCG in future studies of in vivo trafficking experimental and clinical immunotherapy.