The mouse B16 melanoma metastasizes first to the lungs and secondarily to systemic sites, involving mainly the adrenals, ovaries and pancreas. Systemic colonization effected by intracardiac injection of tumor cells establishes similar patterning, but in addition frequently colonizes the bones. To assess possible systemic site influences on metastasis and colony formation, the capacity of B16 melanoma cells to proliferate in these sites in vivo and in ex vivo explants following intracardiac injection was examined. Effects of cells isolated from these sites, and of organ- or tissue-conditioned medium, on growth of B16 cells in monolayer culture were also studied. Injected fluorochrome-labeled tumor cells initially distributed without site preference, but within 48 h had begun proliferating in the adrenals, ovaries and lungs, while remaining static in the pancreas and bones, and disappearing from the spleen, liver, kidneys, brain, and skeletal muscles. Mitogenic activity releasable in soluble form was associated with all favorable organs and tissues and was the predominant influence of those tissues on cultured tumor cells. In contrast, the overall effects of liver, spleen, kidney, and brain tissues were to inhibit tumor cell growth. Soluble growth-promoting activity enhanced clonogenic growth of isolated tumor cells stimulated by mouse serum, suggesting that metastasis or colony formation might be stimulated in favorable sites by those factors together with blood-borne growth factors. The observed effects of organ- and tissue-derived cells and soluble factors on tumor cells generally reflected the in vivo consequences of tumor cell entrapment in the corresponding sites. However, the failure of metastases to develop in the bones, which are favorable sites for colonization by the same cells, remains puzzling.