Catheter recordings of accessory pathway (AP) activation were used to identify the site of antegrade and retrograde AP conduction block in 126 consecutive patients undergoing electrophysiological testing. Activation was recorded from 89 of 121 left free-wall and posteroseptal pathways (left APs) and from 12 of 24 right free-wall, midseptal, and anteroseptal pathways (right APs). The recorded APs were further subdivided into those exhibiting consistent antegrade conduction during sinus rhythm (overt APs: 50 left APs, eight right APs), those exhibiting intermittent antegrade conduction (intermittent APs: six left APs, two right APs), and those exhibiting only retrograde conduction (concealed APs: 33 left APs, two right APs). The sites of block were recorded during decremental atrial and ventricular stimulation. The sites of both antegrade and retrograde block were determined in 40 of 50 overt left APs and six of eight overt right APs. Antegrade and retrograde block occurred at or near the AP-ventricular (AP-V) interface in 37 of 40 overt left APs and two of six overt right APs and at the atrial-AP (A-AP) interface in one of 40 overt left APs and four of six overt right APs. In three of three overt left APs with no retrograde conduction, retrograde block occurred at or near the AP-V interface. The site of antegrade and retrograde block differed in only two of 58 overt pathways. There was no difference between overt APs limited at the A-AP or the AP-V interface in the shortest atrial or ventricular pacing cycle length maintaining 1:1 antegrade or retrograde AP conduction, respectively. Both antegrade and retrograde block occurred near the AP-V interface in four of six intermittent left APs and zero of two intermittent right APs and near the A-AP interface in two of six intermittent left APs and one of two intermittent right APs. The sites of both antegrade and retrograde block were determined in 28 of 33 concealed left APs, and both occurred at or near the AP-V interface in 26 and A-AP interface in two APs. In two of two concealed right APs, antegrade block occurred at the AP-V interface. These findings suggest that both antegrade and retrograde conduction are limited by factors operating near the AP-V interface in overt left APs and at the A-AP or AP-V interface in overt right APs. Factors limiting antegrade conduction in concealed APs appear to be located almost always near the AP-V interface.