TMS pulses over the hand area of motor cortex activate different subpopulations of synaptic connections if the direction of the induced current in the brain is reversed from posterior-anterior (PA) direction to anterior-posterior (AP). We tested whether this also made a difference to the after-effects of paired associative stimulation (PAS: ulnar nerve stimulation followed 25 ms later by a transcranial magnetic stimulation pulse). If 50 pairs of stimuli (0.1 Hz) were applied using conventional suprathreshold PA-PAS in resting subjects, there was no effect on motor-evoked potentials (MEPs) in the first dorsal interosseous muscle. In contrast if the same number of pulses were given while subjects made a small tonic (5% maximum) contraction, MEPs were facilitated and resting motor threshold reduced when AP but not PA pulses were used. Subsequent experiments employed subthreshold TMS (95% of the active motor threshold) during voluntary muscle contraction. MEP facilitation accompanied by reduced AP threshold occurred when PAS was given using AP pulses (AP-Sub-PAS), whereas PAS using PA pulses (PA-Sub-PAS) had no excitatory effect. There was no facilitation if the ulnar nerve stimulus was replaced by digital nerve stimulation. There was a tendency for short interval intracortical inhibition (SICI) to decrease and intracortical facilitation (ICF) to increase after AP-Sub-PAS. We propose that the increased effectiveness of AP-Sub-PAS over PA-Sub-PAS is due to the fact that AP TMS more readily activates I3 inputs to corticospinal neurons and hence that these are an important component of associative plasticity in the human motor cortex.