Hamster cells, transformed in vitro by SV40, have been reported to secrete an unidentified factor(s) that inhibits thymidine uptake (TU) by various normal cell types, including activated lymphocytes. It has been postulated that this apparent antiproliferative effect may play an in vivo role in the high tumorigenic capacity of SV40-transformed hamster cells. In keeping with this hypothesis, Adenovirus type 2-transformed hamster cells, which are only weakly tumorigenic, do not inhibit TU by indicator cells in vitro. To study the biological relevance of this phenomenon, we assayed 11 cell lines derived from different fibrosarcomas, induced in Syrian hamsters by SV40, for their ability to inhibit TU by normal rabbit kidney indicator cells. In contrast to cells transformed in vitro by SV40, media conditioned by 6 of 11 tumor-derived cell lines did not inhibit TU. Our results do not support the hypothesis that an antiproliferative factor secreted by SV40-transformed cells promotes the tumor-inducing capacity of these cells. Furthermore, inhibition of TU does not appear to be due to the production of a specific antimitotic peptide, but rather to other biochemical properties of the media conditioned by transformed cells. Finally, these biochemical properties do appear to correlate with specific morphological and growth characteristics of the tumor cells, but probably as an effect and not a cause.