A complex is formed upon incubation of a precursor mRNA (pre-mRNA) with HeLA cell nuclear extract in the absence of added ATP (-ATP complex). Pre-mRNAs with mutations in the 5' splice site, the 3' splice site, or the polypyrimidine tract did not form this complex. Once formed, the -ATP complex was stable to competition by excess pre-mRNA. The complex was shown to contain the U2 small nuclear ribonucleoprotein particle (snRNP) and was distinct from the previously described U2 snRNP/pre-mRNA complex, the prespliceosome. These complexes have different electrophoretic mobilities, ATP requirements, and sensitivities to mutations of the 5' splice site. Although U1 snRNP was not found in the -ATP complex, a requirement for the U1 snRNP was suggested by immunodepletion experiments. The possible implications for the study of spliceosome formation are discussed.