Most biological effects of tissue plasminogen activator (tPA), such as fibrinolysis, are mediated by its protease activity. Recent studies, however, have demonstrated that tPA also has several protease-independent effects such as: neuroprotection, microglial activation, and promoting LTP formation. In order to gain a better understanding of how tPA affects neurons, we examined neurite outgrowth and cell survival in low density cerebrocortical neuronal culture in the presence of tPA. tPA enhanced neurite elongation and neuronal survival. tPA protease inhibitors, PAI-1 or PMSF, did not alter either effect. Consistent with neurotrophic effects, tPA activated Raf-K/ERK, PKC and PI3-K/Akt, 5-60 min after treatment. In addition, specific inhibitors of these kinases reduced tPA-induced neurite outgrowth. Interestingly, survival-promoting effect of tPA was attenuated only by PI3-K inhibitors. Activation of signaling kinases suggests that tPA activates an upstream membrane receptor. Thus far, three membrane proteins, low density lipoprotein receptor-related protein (LRP), mannose receptor (MR), and annexin-II (AII), have been identified to bind tPA. While inhibiting LRP or MR did not change tPA-induced neurite outgrowth and cell survival, inhibiting AII blocked neurotrophic effects of tPA. Taken together, our results indicate that tPA has novel, non-proteolytic neurotrophic effects on cultured cortical neurons, which are likely mediated by AII.