The immunosuppressive macrolide FK-506 has been shown to protect neurons in culture against glutamate excitotoxicity. This effect was attributed to the binding of immunosuppressants to calcineurin-inhibiting immunophilins. We now report that also the non-immunosuppressive macrolide antibiotics protect neurons in culture against NMDA- but not kainate-mediated excitotoxicity. The effect was structure-dependent: larger macrolide rings were more active. Macrolides did not affect the 3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid (CPP) binding or the NMDA-mediated calcium influx.