Long-term depression (LTD) was induced in rat cerebellar slices by conjunctive stimulation of parallel fibers (PFs) and climbing fibers (CFs) under perfusion of 20 microM picrotoxin. LTD was estimated by the reduction in the initial rising slope of EPSPs PF-induced in Purkinje cell dendrites. LTD-inducing efficacy was represented by both the average amount of depression and the probability of inducing depression greater than 25%, both measured at 40 min after the onset of conjunctive stimulation. Using 300 regularly recurring pulses given to both CFs and PFs with 0 ms interval, LTD was optimally induced at 1 Hz, and to lesser degrees at other frequencies. When the number of conjunctive stimuli at 1 Hz with zero CF-PF interval was varied from 50 to 500, 300 stimuli induced LTD most robustly. When CF-PF interval was varied while 300 pulses were given at 0.25-4 Hz, LTD was induced even when PF stimuli were delayed after CF stimuli by as much as 2 s, but it was inhibited when PF stimuli preceded CF stimuli by 10-100 ms. LTD was also induced by applying repeated short pulse trains to both CFs and PFs, but repeated application of a PF stimulus train immediately followed by a CF stimulus train as in classical conditioning was effectless. The present results suggest complex processes leading to LTD as a result of conjunctive CF and PF stimulation.