Atrial fibrillation (AF) catheter ablation is performed in patients receiving direct oral anticoagulants (DOACs). Guidelines recommend to perform the procedure with uninterrupted anticoagulation and intra-procedural unfractionated heparin (UFH) administration to achieve activated clotting time (ACT) at 300 seconds, as recommended for vitamin K antagonist (VKA). To determine whether ACT monitoring might be transposed from VKA to DOAC-treated patients. From 124 patients receiving either uninterrupted DOAC (dabigatran, rivaroxaban, apixaban) or VKA or being untreated, blood sampling was performed for anticoagulation testing. DOAC concentrations, and INR for VKA were measured. ACT was determined at baseline, and after spiking with UFH doses equivalent to 1000, 2500, 5000 and 10000 IU in vivo. At baseline, the extent to which oral anticoagulants prolonged ACT differed: despite similar concentrations, ACT was longer with dabigatran and shorter with apixaban (both P < 0.001). The relationship between ACT and the intensity of anticoagulation also differed: ACT strongly correlated with INR ( r = 0.73, P < 0.001) and dabigatran concentration ( r = 0.87, P < 0.0001), but poorly correlated with apixaban or rivaroxaban concentrations. Moreover, the effects of UFH on ACT prolongation depended on the anticoagulant onboard: dose-response curves in samples from VKA and dabigatran-treated patients were parallel whereas ACT prolongation in response to UFH was significantly lower in the presence of FXa-inhibitors, especially apixaban. UFH administration to achieve ACT at 300 seconds may be transposed from VKA to uninterrupted dabigatran-treated patients but not to patients receiving FXa inhibitors, especially apixaban: targeting 300 seconds might expose to UFH overdosing, and bleeding complications. These findings question the indiscriminate use of ACT in AF ablation.