Untransformed, non-tumorigenic mouse cells respond to cytochalasin B (CB) with limited nuclear division. BALB/c mouse embryo fibroblasts (MEF) and both BALB/c 3T3 and Swiss 3T3 cells become binucleated in the presence of CB and cells with three or more nuclei are very rare or undetectable. MEF are diploid (40 chromosomes) and 3T3 cells are subtetraploid (74-76 chromosomes). Transformation of MEF by SV40 produces a dramatic change in response to CB. These cells, which contain SV40 T-antigen, become highly multinucleated in the presence of CB. More than 20% of the cells have at least seven nuclei. Also premature chromosome condensation (PCC), an abnormality rare in CB-treated normal cells but one which is common to highly multinucleated cells, is frequent and occurs in at least 10% of mitoses. SV40-transformed MEF have 40 or 80 chromosomes, e.g. are diploid or tetraploid. Transformation of 3T3 by SV40 or adenovirus type 12 does not result in a marked change in the response to CB. Although some trinucleate and tetranucleate cells are formed, cells with more nuclei are undetectable or rare. PCC is also rare. These cells show chromosome numbers somewhat lower than their untransformed parents and in one line the chromosome number appears to decrease with passage of the cells. This failure to undergo a marked change in responsiveness to CB following transformation is not a characteristic of all transformed 3T3 cells. SVT2, a line of 3T3 which was transformed by SV40 prior to its establishment as a continuous line, responds to CB with a high degree of multinucleation. These cells are diploid. These results suggests that 3T3 cells are constitutive for controlled nuclear division and that this feature may be related to chromosome constitution.