We compared the mitogenic response of Schwann cells freshly isolated from adult, neonatal, and embryonic nerves, and compared these cells with cells that had been cultured in serum for 5 days. DNA synthesis in response to growth factors was measured using bromodeoxyuridine and immunocytochemistry. Freshly isolated adult Schwann cells were unresponsive to growth factors with or without forskolin to elevate intracellular cAMP levels. After 5 days of culture in serum, or alternatively in defined medium containing fibroblast growth factor 2 plus forskolin, or neu-differentiation factor beta2, adult cells were responsive to mitogens, whereas cells cultured in defined medium alone remained unresponsive. Serum also increased expression of type 1 fibroblast growth factor receptor. Freshly isolated embryonic and neonatal Schwann cells in contrast responded to growth factors even in the absence of forskolin. This responsiveness changed with time in culture. Neonatal cells cultured for 5 days in defined medium in the presence or absence of serum no longer responded to FGF alone, but required forskolin for a mitogenic response. Thus, the response of freshly isolated cells to mitogens is developmentally regulated; extrinsic signals are required to render adult cells responsive to mitogens; and with time in culture, neonatal cells develop a requirement for cAMP elevation for mitogenic response.