Results are reported showing that pituitary extracts stimulate growth of 3T3 cells, an established line of mouse fibroblast. Cells were cultured in medium containing an organic buffer (HEPES; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), which increases the serum requirement for cell growth. The pituitary growth-prompting activity is (a) not dialyzable; (b) thermolabile; (c) protease sensitive; and (d) enhanced by hydrocortisone (a steroid hormone). Activity was quantitatively assayed by measurement of stimulation of cell division or [(3)H]thymidine uptake into DNA. The active species is probably different from pituitary protein hormones and it seems to be effective at concentrations observed for protein hormones in vivo. 3T3 cells transformed by simian virus 40 are not responsive to this activity.