Homogenates of human testes, epididymides and prostate, and calf testes and epididymides are mitogenic for cultured human foreskin fibroblasts. The growth factors appear similar in that they are inactivated by boiling and acid, but not by treatment with reducing agent. The growth factor in human and bovine testes was partially purified from tissue homogenates, prepared in high ionic strength buffer (pH 7.6) containing protease inhibitors, by ammonium sulfate precipitation and two cycles of heparin-Sepharose chromatography. The growth factor in calf testes was also partially purified from tissue extracted in ammonium sulfate without protease inhibitors, acidified to pH 4.5, and precipitated by ammonium sulfate followed by two cycles of heparin-affinity chromatography. A predominant 17,500 molecular weight (MW) growth factor was identified from alkaline homogenates of human and calf testes by its reactivity with antisera prepared against synthetic peptides whose sequences corresponded to residues 1-12 (amino-terminal), 33-43 (internal) and 136-145 (carboxy-terminal) of bovine basic fibroblast growth factor (bFGF). A slightly smaller 16,600 MW peptide from acidic extracts of calf testes also reacted with antisera to the three synthetic peptides. A 15,500 MW peptide, lacking immunoreactivity with antiserum to the amino-terminal synthetic peptide, was also seen. These findings suggest that a growth factor is present in human and calf testes that is structurally related to bFGF. The structure of the growth factors appears to be altered during the isolation procedure.