Intracortical inhibition was investigated in normal human volunteers by paired-pulse transcranial magnetic stimulation, using a new, computer-assisted threshold-tracking method. Motor threshold was defined as the stimulus amplitude required to evoke a motor evoked potential of 0.2 mV (peak-to-peak) in abductor pollicis brevis, and inhibition was measured as the percentage increase in threshold, when the test stimulus was preceded by a subthreshold conditioning stimulus. This method was used to investigate the dependence of intracortical inhibition on conditioning stimulus parameters and on voluntary activity. Interstimulus interval (ISI) was first stepped from 1 to 4.5 ms, as inhibition was measured using conditioning stimuli of fixed amplitude (50-90% resting motor threshold). Maximal inhibition was produced at ISIs of 1 and 2.5 ms. The effect of conditioning stimulus intensity was then assessed at these ISIs. Inhibition occurred at significantly lower conditioning stimulus intensities with ISI=1 ms than with ISI=2.5 ms. Voluntary activity reduced inhibition at both ISIs, but had a much greater effect on inhibition at ISI=2.5 ms. Inhibition during voluntary activity was also examined for single motor units in first dorsal interosseous by generating poststimulus time histograms. Inhibition, indicated by a reduction in the later peaks of increased firing, was observed with ISI=1 ms, but not with ISI=2.5 ms. We conclude that there are two distinct phases of inhibition, occurring at ISI=1 ms and ISI=2.5 ms, differing both in thresholds and susceptibility to voluntary activity.