Tumor cell traffic between intramuscular tumors experimentally induced in nude mice and lymph nodes was studied using PC-3.luc prostate adenocarcinoma cells permanently transfected with the luciferase gene as a tumor cell marker. This sensitive approach allowed the detection of 1 luminescent tumor cell mixed with 1 x 10(7) unlabeled PC-3 cells and of 1 tumor cell/lymph node. PC-3.luc cells inoculated in nude mice showed a 1000-fold expansion, accompanied by a 4.5-fold increase in tumor cell density (tumor cell number/gram of tumor), during the first 90 days of primary tumor growth. No macroscopic secondary tumors were found in organs, other than lymph nodes, by the end of the experiment. Tumor cell spread to lymph nodes was detected at Day 21, when there were 2 x 10(5) tumor cells at the inoculation sites, before discrete primary tumors could be identified. The total tumor cell burden in the tested lymph nodes was modeled by a power function of primary tumor cell number (determination coefficient R2 = 0.9472). By the end of the experiment, on Day 110, there were 1.8 metastatic cells in the studied lymph nodes for every 1000 primary tumor cells. These results suggest that empirically obtained tumor-specific indexes could be used to characterize the invasion of lymph nodes by tumor cells. The path of spread for PC-3.luc cells from intramuscular sites appears to follow the lymphatic system, and at no time during the experiment were tumor cells found in blood. An upper limit of no more than 16 blood-circulating tumor cells was established for these experiments. The observation of tumor cells that were invading the lymphatic system from the onset of tumor growth but unable to establish secondary tumors in other organs emphasizes the potential of this procedure in studying the multi-step nature of metastasis.