To quantitatively characterize flecainide-induced channel blockade from usedependent conduction delay (CD), 12 canine purkinje fibers were studied using a dual microelectrode technique. During 60 sec of pacing at interstimulus intervals (ISI) of 1.25–0.4 sec with 2 μ M flecainide (FLEC), incremental CD followed a monoexponential time course, the rates of which were linearly related to the interpulse recovery interval (t r = ISI — action potential duration). Steady state block was an exponential function of the recovery rates. Use-dependent block derived from incremental CD and decremental squared conduction velocity ( θ 2 ) was characterized by the forward (k) and reverse (I) rate constants for the activated (a) and resting (r) states: k a (× 10 6 ) (mol -1 s -1 ) l a (s -1 ) k r (× 10 2 ) (mol -1 s -1 ) I r (s -1 ) CD 7.0 ± 2.6 12.0 ± 4.4 0.6 ± 1.7 4.01 ± 1.63 θ 2 10.0 ± 3.4 14.7 ± 2.5 2.8 ± 5.7 3.66 ± 1.40 V max(prox) 6.8 ± 2.3 15.9 ± 5.0 5.1 ± 10.3 4.22 ± 1.11 These rates reflect marked open state Na+ channel block and closed channel trapping at resting membrane potentials with FLEC. The addition of 1 μ M isoproterenol (ISO) to FLEC-superfused fibers reversed the FLEC-induced reduction of θ 2 from 1.79 ± 0.7 to 1.89 ± 0.89 (m/s) 2 (p = 0.017) without changing V ˙ max . The rate constants for FLEC binding and unbinding were not altered by ISO. Thus FLEC's apparent binding rates can be quantified from its use-{jependent effects on conduction. Both ISO's selective reversal of FLEC effect on θ 2 but not V ˙ max and the absence of changes in the rate constants suggest that the modulation of FLEC effect is due to an alteration in passive membrane properties. These characterizations will facilitate subsequent comparisons of FLEC interactions in pathologic and hyperadrenergic states in vivo .