Cell proliferation is a key primary process during neural development and also plays an important role in the regenerative response of neural tissue to injury. It has been reported that glial cell proliferation is, at least in part, controlled by a neuronal signal, possibly involving cell surface molecules. We report here that the addition of purified rat neural cell adhesion molecule (N-CAM) to primary cultures of rat forebrain astrocytes inhibits their proliferation. This inhibitory effect can be elicited in cultures grown in chemically defined serum-free medium or in medium that had been supplemented with growth factors. Polyclonal antibodies to N-CAM or their Fab' fragments elicited a similar inhibitory effect. The magnitude of the inhibitory effect of N-CAM was dependent on cell density: it was maximal at low cell densities and weakened progressively as cells approached confluency. Synthetic peptides with sequences identical to a putative homophilic binding region of N-CAM mimicked the effect of purified N-CAM, while peptides of the same length and amino acid composition but with a randomized sequence did not. The addition of N-CAM antisense oligonucleotides to primary astrocyte cultures for 48 h resulted in reduced levels of N-CAM expression. After N-CAM levels on astrocytes were diminished by this treatment, the antiproliferative effect of N-CAM added to the medium was significantly reduced. The combined results suggest that N-CAM homophilic binding may be involved in the control of glial cell proliferation.