In the experimental model system where human tumor cells (HEp3) are implanted on the chorioallantoic membrane (CAM) of the chick embryo, metastasis of HEp3 cells to the embryonic lung occurs within a few days. Such rapidity in tumor dissemination makes this an attractive and potentially useful model for studying the metastatic process. The model, however, involves microvascular trauma at the site of implantation and thus tumor cells may accidentally enter the circulation during implantation or shortly thereafter. If these cells are the cause of the lung metastasis subsequently measured, the model would be in effect a colonization system and not a true, spontaneous metastasis system. The possible contribution of accidental lung colonization to secondary tumor growth was therefore critically examined in this model. In standard metastasis assays, HEp3 was inoculated onto the CAMs of 10-day embryos, which were then incubated for various periods of time. The embryos' lungs were passaged to a second group of CAMs, incubated for 7 days to allow expansion of any HEp3 cells present, and then assayed for HEp3 cells by both microscopy and measurement of human plasminogen activator (PA) activity. Metastasis was evidenced by PA values above background (30 mU/mg protein). Morphological analysis of HEp3 cells in the embryonic lung correlated closely with PA values. To focus on the early stages of tumor dissemination when colonization might occur, the primary tumor was surgically excised from 38 embryos at various intervals after tumor inoculation, and after the operation embryos were allowed to develop to day 17. This procedure increased estimated assay sensitivity down to the level of 1 to 10 cells per lung in embryos operated on within 2 days of inoculation. Median PA values in the transplanted lungs were 13, 3, 37, 1,290 and 3,765 mU/mg protein in the groups operated on at 4 hr, 1, 2, 3 and 4 days after inoculation, respectively. Thus very few or no HEp3 cells arrest and grow in the lungs during the first 24 to 48 hr, but extensive metastasis occurs by 72-96 hr. Accidental colonization therefore plays no major part in the rapid pulmonary spread of HEp3 in this model.