The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits N-methyl-d-aspartate (NMDA) receptors through a direct effect. Whole-cell NMDA-activated current was recorded in native receptors from mouse hippocampal slice culture and rat recombinant NR1aNR2A and NR1aNR2B receptors transiently expressed in HEK293 cells. Extracellular application of genistein and NMDA reversibly inhibited NMDA-activated current. The inhibition of NMDA-activated current by genistein applied externally was not affected when genistein was also pre-equilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited NMDA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the NMDA response. Moreover, genistein-induced inhibition of NMDA-activated current displayed concentration- and voltage-dependence. Our results demonstrate that genistein has a direct inhibitory effect on NMDA receptors that is not mediated via inhibition of tyrosine kinase. Thus, other PTK inhibitors may be more suitable for studying involvement of PTKs in NMDA receptor-mediated events.