Swiss 3T3 cells, starved to quiescence in low serum concentration, initiate DNA synthesis and undergo mitosis in low serum concentration (less than 0.1%) after a short exposure (2-10 min) to alkaline medium (pH 8.5-10). This study shows that initiation of DNA synthesis and mitosis can be dissociated from growth in cell size. Alkaline-stimulated cells do not increase in size before mitosis (unbalanced growth) in contrast to quiescent cells stimulated by 10% serum, which approximately double their protein content before mitosis (balanced growth). The exposure to the alkaline medium per se does not render the cells incapable of growing in size, since alkaline-treated cells, which are subsequently cultured in 10% serum, undergo balanced growth to the same extent as cells only stimulated by 10% serum without alkaline pretreatment. This study also shows that the small proportion of cells undergoing background proliferation in low serum concentration (less than 0.1%) do not increase in size before mitosis. The extent to which alkaline-stimulated cells, as well as cells undergoing background proliferation, grow in size before mitosis, is correlated to the serum concentration in the culture medium (DMEM). In 0.1% serum or less the cells do not grow in size. In 0.5% serum the cells increase in size by 30% before mitosis and complete balanced growth is achieved in medium containing 2% serum or more. These findings indicate that some macromolecular factor or factors in serum are required for growth in cell size before mitosis.