Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [3H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [3H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [3H] thymiding incorporation into DNA. MSA causes a 2--10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [3H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [3H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.