The rate and level of classical nictitating membrane (NM)/eyelid conditioning in rabbits established by pairing a pontine nucleus stimulation conditioned stimulus (CS) with an air puff unconditioned stimulus (US) were studied at 6 interstimulus intervals (ISIs). Similar to earlier studies which used peripheral CSs, an inverted U-shaped function relating ISI and conditioning was generated. Interstimulus intervals of 250 and 500 ms produced the highest levels of conditioning, 100, 1000 and 2000 ms ISIs resulted in lower levels of conditioning, and no conditioning was established with a 50 ms ISI. These results demonstrate that a normal ISI function can be established when direct activation of cerebellar mossy fibers is used as a CS instead of conventional peripheral CSs.