We studied the factors that control the capacity of tumor cells to grow in soft agar. And, we analyzed the effect of cell-free ascites (CFA) obtained from ovarian cancer patients in combination with various media on the cloning efficiency of H-134 and OVCAR-3nu ovarian carcinoma cell lines and the WiDr colon carcinoma cell line. Seven batches of CFA consistently enhanced the soft agar growth of tumor cells more efficiently than tested sera. The addition of charcoal-treated bovine serum albumin (BSA) lowered the amount of CFA required for optimal tumor cell growth. As little as 1.25 ng of epidermal growth factor (EGF)/ml further improved OVCAR-3nu soft agar growth in combination with all of the amounts of CFA tested. Thus, neither BSA nor EGF seems to account for the colony-stimulating effect of ascites on tumor cells. Four batches of CFA were tested for stimulating soft agar growth of normal rat kidney (NRK-49F) fibroblasts; all induced colonies of different morphologies. This effect was potentiated by EGF, which suggests the presence of several transforming growth factor-like activities in CFA. The results show that differences in cloning efficiency of tumor cells of one or two orders of magnitude can be found between standard (anchorage-dependent growth-supporting) media and media optimalized for soft agar growth, such as CFA in the presence of EGF. This paper will discuss the similarity in effects of CFA on various tumor cells and NRK cells, and possible implications of the stimulatory effects of CFA.