A variety of agents can induce mammalian tumor cell lines to acquire characteristics of the normal cell counterpart. Dimethylsulfoxide (DMSO) has been an effective differentiating agent in many tumor cell lines. In the present study a Dunning rat prostate tumor subline, MAT LyLu, available as an in vitro continuous cell culture was treated with 2.25% DMSO (vol/vol). Treated MAT LyLu cells had a decreased growth rate, saturation density, and clonogenicity, an increased doubling time, and alterations in enzyme activity and tumorigenicity when compared to untreated MAT LyLu cells. The cell viability of treated cells at the saturation density was greater than 90%. MAT LyLu cells treated with DMSO and then removed from DMSO (posttreated) when compared to untreated cells had similar growth rates, doubling times, clonogenicities, enzyme activities, and tumorigenicities. Posttreated MAT LyLu cells had a different growth pattern than untreated MAT LyLu cells. Posttreated cell viability at saturation density was greater than 90%. This investigation demonstrated that a rat prostate adenocarcinoma grown in medium containing 2.25% DMSO acquired characteristics consistent with differentiated prostate cells. Posttreated MAT LyLu cells were similar in many characteristics to untreated cells but were not identical. The alterations noted were not cytotoxic and were not completely reversible. The results of this study correlated with the observations of other investigators who have studied mammalian tumor cell lines exposed to DMSO.