Bovine milk contains growth promoting factors that stimulate DNA synthesis and cell division in confluent monolayers of quiescent Balb/c 3T3 cells. The growth factor activity was highest in colostrum obtained within 24 hours after birth of a calf. Samples of milk obtained 32 hours and 60 hours after birth were 20% and 1% as active respectively as was a sample obtained 8 hours after birth in stimulating DNA synthesis. No activity was detectable 3 days after birth or thereafter. A similar temporal dependence was found in sheep's milk. Bovine colostrum obtained on the day of a calf's birth can be substituted for serum and will support the growth of sparse Balb/c 3T3 cells to confluence. In Dulbecco's modified Eagles's medium (DMEM) supplemented with 2.5% (vol/vol) bovine colostrum, the number of Balb/c 3T3 cells in a dish increased 35-fold, from 2.0 X 10(4) cells to 7 X 10(5) cells. The generation time was approximately 38 hours. Proliferation of cells was characterized by formation of clusters of confluent Balb/c 3T3 cells which were smaller in size and more tightly packed than were Balb/c 3T3 cells grown to confluence in serum. No proliferation was detected in DMEM supplemented with milk obtained 10 days after birth of a calf or in DMEM supplemented with bovine serum albumen.