Hamster BHK21 tumor cells and human rhabdomyosarcoma (RD) cells responded to cytochalasin B (CB) by becoming highly multinucleated. Nearly 80% of the BHK cells contained four or more nuclei after 7 days of exposure to CB, and 35% of these had at least seven nuclei. Similar frequencies of multinucleation were obtained with RD cells. Administration of 10(-3) M theophylline and dibutyryl adenosine 3',5'-cyclic monophosphate (Bu2cAMP) at 5X10(-4) M or 10(-3) M to CB-treated RD or BHK cells greatly reduced the frequency of cells with five or more nuclei. The frequency of cells with seven or more nuclei was reduced to less than 5%. Along with this reduction in highly multinucleated cells was an increase in the incidence of binucleated cells. Bu2cAMP was toxic and caused many CB-treated BHK cells to detach from the culture surface, but not all cells were killed. Bu2cAMP had little toxic effect on CB-treated RD cells. These observations indicated that the inhibition of high degrees of multinucleation were not the result of nonspecific toxic effects of Bu2cAMP but that nuclear division was limited by it. The effect of Bu2cAMP on density-dependent inhibition of growth was also studied. Addition of only theophylline and Bu2cAMP to either BHK or RD cells resulted in growth to significantly lower saturation densities. The toxicity of Bu2cAMP on cells in crowded cultures apparently caused the limited propagation. Bu2cAMP resulted in significant cell killing or detachment but, once the lower saturation densities were reached, cell death was minimized. Thus Bu2cAMP did not restore contact inhibition per se. It was also found that untreated RD cells grew to lower concentration densities than expected from the microscopic inspection of cells in situ. Microscopic inspection revealed high concentration densities and numerous mitoses. This apparent contradiction was due to the ability of RD cells to fuse upon the attainment of confluence and to produce multinucleated cells without the aid of CB.