The nature of open state block was characterized in isolated canine cardiac sarcoplasmic reticulum (SR) potassium channel incorporated into planar lipid bilayers. 4-Aminopyridine (4-AP) blocked the open conductance state of the potassium channels in a voltage-dependent manner. Blockade was reversible, occurred from either the cis (cytoplasmic) or the trans (lumenal) side and was competitive with potassium ions. Reversal potential measurements indicated that this channel was impermeable to 4-AP. Measured effective electrical distances were roughly symmetrical and indicated penetration of 0.39 and 0.42 of the membrane electrical field from the cis and trans sides, respectively. Effective electrical distance was insensitive to potassium ion concentration in the range 50 to 200 mM and indicated that 4-AP was able to penetrate relatively deeply into the pore compared with blockade of sarcolemmal potassium channels. Potassium ion concentration and voltage dependence of 4-AP blockade were consistent with a two binding site blockade model, similar to the model used previously to describe calcium ion blockade of the SR potassium ion channel. Unlike calcium blockade, however, 4-AP blocked from either cis or trans in a similar manner, suggesting a distinct binding site for each of these two blockers. Open channel, voltage-dependent blockade of the SR potassium channel by 4-AP is in marked contrast to its action on sarcolemmal potassium channels and suggests that either 4-AP penetrates much farther into the potassium channel permeation pathway than was previously believed, or the SR potassium channel has a very different physical pore arrangement from that of sarcolemmal potassium channels.