Primary cultures of bovine calf anterior pituitary cells were observed to proliferate for at least eight doublings in a serum-free defined medium without the addition of mitogens or hormones. Insulin caused minor increments both in the rate of proliferation of cells from a doubling time of 31 h to 26 h and in the saturation density from 1 X 10(5) to 1.4 X 10(5) cells/cm2. To determine whether the pituitary cells could secrete a growth factor, medium conditioned by the pituitary cells was tested for mitogenic activity. Conditioned medium caused a dose-dependent increase in 3T3 fibroblast DNA synthesis indicating that it contained the equivalent mitogenic activity of either 2.5 X 10(-9) M epidermal growth factor (EGF) or 250 ng/ml fibroblast growth factor (FGF). 3T3 cell proliferation was also stimulated to a greater extent by conditioned medium than by EGF or FGF. Y1 adrenal cortical tumor cells were also stimulated by conditioned medium to synthesize DNA and proliferate. The mitogenic activity in the conditioned medium was heat and acid stable unlike pituitary FGF. No EGF could be detected in the conditioned medium by a RIA using antimouse EGF serum and a RIA for multiplication-stimulating activity was also negative. None of the classical pituitary hormones were mitogenic under the test conditions indicating that the mitogenic activity could not be attributed to the known pituitary hormones. Although these results indicate that pituitary cells secrete a growth factor(s), its identity is not established.