The growth of axons is fundamental to the development and repair of brain circuitry. We show here that Mst3b, a neuron-specific homolog of the yeast kinase Ste20, is critical for axon outgrowth. Mst3b is activated in response to trophic factors, and suppressing its expression (via siRNAs) or its function (by a dominant-negative mutant) blocks axon outgrowth. Inosine, a purine nucleoside that stimulates axon outgrowth, activates Mst3b kinase activity, whereas 6-thioguanine, a purine analog that blocks outgrowth, inhibits the activity of this kinase. These findings place Mst3b as a key regulator of axon outgrowth and help explain the purine sensitivity of this process.A xon outgrowth begins shortly after neurons undergo final cell division, and it ends when axon terminals reach their appropriate targets. Although this process is not normally reactivated in the mature CNS after injury, some degree of CNS regeneration has been achieved experimentally by altering neurons' intrinsic growth state and by counteracting inhibitory signals associated with myelin, the perineuronal net, or a glial scar (1-5). Thus, understanding the mechanisms that underlie axon growth not only affords insight into how the brain's circuitry develops but may also enable us to improve functional outcome after stroke, trauma, or neurodegenerative disorders.The purine nucleoside inosine stimulates axon outgrowth in certain types of neurons in culture and in vivo after CNS injury (6-10). The purine analog 6-thioguanine (6-TG), on the other hand, blocks outgrowth induced by neurotrophic factors (9,11,12), and this effect is paralleled by the inhibition of a previously unidentified 45-50 kDa serine-threonine protein kinase (13). Inosine competitively reverses the inhibitory effects of 6-TG on outgrowth (7, 9), suggesting that it may act as an agonist of the 6-TG-sensitive kinase. We now identify the purine-sensitive kinase as mammalian Ste20-like protein kinase-3b (Mst3b) and show that it is a key regulator of axon outgrowth.