Extracts from bovine seminal vesicles have been shown to contain high concentrations of nerve growth factor (NGF)-like biological activity and of the NGF protein with properties corresponding to that of NGF from other sources. We now demonstrate that a second neuronotrophic protein, termed seminal vesicle-derived neuronotrophic factor (SVNF), is present in seminal vesicle extracts (SVEs), which could not be distinguished from NGF on the basis of biological activity. SVNF has neuronotrophic activity on NGF target cells like embryonic chicken-sensory and sympathetic neurons, sympathetic neurons, and chromaffin cells from neonatal rats, but it is inactive on embryonic chicken ciliary or neonatal rat nodose ganglion neurons. It also stimulates fiber outgrowth from rat pheochromocytoma (PC 12) cells. In gel filtration chromatography on Biogel A 1.5 m, the activity is eluted with an apparent molecular weight of 40 kilodaltons, and by preparative isoelectric focusing, the isoelectric point was determined to be in the neutral range (6.8-7.8). The biological activity of SVNF, in contrast to that of NGF, is partially retained after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and can be electrophoretically eluted with an apparent molecular weight of 16-20 kilodaltons. Electrophoretically purified SVNF is not inhibited by antisera to mouse NGF, but its activity is increased greater than 10-fold in the presence of very low concentrations of NGF. For partially purified SVNF, a specific activity of 2.9-5.8 X 10(5) biological units/mg of protein was determined in the presence of subthreshold NGF concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)