We describe the isolation and biochemical characterization of [U4/U6.U5] tri-snRNP complexes from HeLa cells under nondenaturing conditions using a monoclonal antibody reacting with the U5-specific 100-kD protein. We show that the [U4/U6.U5] complex contains five previously unobserved proteins with molecular masses of 90, 60, 27, 20, and 15.5 kD, in addition to the core proteins, common to the U4/U6, U5, U1, and U2 snRNPs, and the U5-specific proteins, as found in 20S U5 snRNPs. With approximately 20 distinct snRNP proteins the complexity of the [U4/U6.U5] tri-snRNP is surprising. One or more of the five proteins found exclusively in the 25S [U4/U6.U5] tri-snRNP appears to be involved in the assembly of the tri-snRNP complex, as, in an in vitro reconstitution assay, purified 20S U5 and 10S U4/U6 snRNPs formed stable 25S [U4/U6.U5] complexes only in the presence of the free tri-snRNP-specific proteins. The formation of the [U4/U6.U5] complex in vitro does not require ATP, and the stability of the purified tri-snRNP complex is not affected by ATP to a measurable extent. However, the native [U4/U6.U5] displays a kinase activity that is absent in isolated U5: A 52-kD protein present in both U5 and [U4/U6.U5] is phosphorylated only in the latter. The function of this phosphorylation is unclear thus far; it may be involved in the activation of [U4/U6.U5] in the spliceosome.