The major toxic component of black widow spider (Latrodectus mactans tredecimguttatus) venom, alpha-latrotoxin, is known to form ionic channels in different membranes. In order to probe the extramembrane domains of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar lipid membrane were treated with antibodies to latrotoxin or with pronase added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conductance only at positive potentials across the membrane. In contrast, trans side addition of antibodies changed the channel conductance at both positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the initial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and trans application of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, Li+ ions did not change significantly but Cd2+ block was decreased. Trans pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings as evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depends on membrane potential.