If soft agar colony formation assays for primary human tumor cells are going to be performed and the results assessed by optical analysis of colony formation, then we believe it is mandatory in such assay techniques to objectively count control plates on the day of culture initiation using stained plates, use universal cytotoxic control compounds which should uniformly eliminate all viable cell proliferation, and use a vital stain such as the tetrazolium dye INT to document the presence or absence of viable cell colonies when cultures are assessed. There is no question, however, after careful observation of thousands of soft agar cultures of primary human tumor cells in my laboratory, that significant proliferation of small tumor cell aggregates often takes place in vitro and can in fact be used to assess cytotoxic drug effects in vitro. We do not believe that this is demonstrably stem cell or clonal growth. Nevertheless, it almost certainly is in vitro tumor cell proliferation. With very careful controls, we believe that optical methods can be used reliably to evaluate drug effects on this soft agar proliferative capacity of primary human tumor cells. However, it may eventually prove more useful to study human tumor cell proliferation in vitro (even soft agar colony growth) by other methods, such as incorporation of radiolabeled bases into newly synthesized DNA or other macromolecules, or by the simple use of vital dyes, as discussed elsewhere in this volume.(ABSTRACT TRUNCATED AT 250 WORDS)