A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.