A hippocampal astrocyte conditioned medium (HACM) supported the survival of hippocampal neurons under a serum-, glia-free culture setting. The neurotrophic activity in HACM was mostly recovered in low molecular weight fractions (Mr < 3000), which contained high levels of L-serine and L-alanine. However, L-serine alone significantly improved the neuronal survival and neurite growth in a stereo-specific manner. Other non-essential amino acids had no effect. These results strongly suggest that L-serine, released by astrocytes, is essential for the survival and phenotypic growth of hippocampal neurons.