A study has been made to determine the accuracy of the trypan blue staining technique as an index of the proportion of viable cells in a cell population. Eosin and acridine orange were also tested in preliminary studies but were found to be relatively toxic. Cell cultures in vitro and tumor cell populations tested in vivo are used for direct comparisons between viability values obtained by staining cell populations with trypan blue, and values obtained by testing the growth potential of the same cell populations under optical conditions of cultivation. Data are included relative to the effects of trauma, freezing, and of various dispersal agents upon cell viability. The results indicate correlation but not absolute agreement between the two methods (staining and cultivation). Trypan blue values are shown to be 85% (or better) of cultivability values under optimal conditions of handling, but they are grossly inaccurate and unpredictable following traumatic treatment of cell populations. Before trypan blue staining is accepted as an index of cell viability, it is recommended that the specific system under study first be carefully defined.