The mammalian cortex is densely populated by extensively branching, thin unmyelinated axons, which form en passant synapses. Some thin axons in the peripheral nervous system hyperpolarize if action potential frequency exceeds 1-5 Hz. To test the hypothesis that cortical axons also show activity-induced hyperpolarization we used extracellular recording from individual CA3 pyramidal neurons while activating their axon with trains consisting of 30 electrical stimuli. Synaptic excitation was blocked by kynurenic acid. We observed a positive correlation between stimulation strength and the number of consecutive axonal stimuli that resulted in soma spikes, suggesting that the threshold increased as a function of the number of spikes. During trains without response failures there was always a cumulative increase in the soma response latency. Intermittent failures, however, decreased the latency of the following response. At frequencies > 1 Hz, the threshold and latency increases were enhanced by blocking the hyperpolarization-activated H-current by 25 µm ZD7288 or 2 mM Cs⁺. Under these conditions response failures occurred after 15-25 stimuli, independent of the stimulation strength. Adding GABA receptor blockers (saclofen and bicucculine) and a blocker of metabotropic glutamate receptors (MSPG) did not change the activity-induced latency increase in recordings of the compound action potential. We interpret these results as an activity-induced hyperpolarization that is partly counteracted by an H-current. Such a hyperpolarization may influence transmitter release and conduction reliability of these axons.