Northern blot analysis of HeLa cell nuclear extract following electrophoresis in nondenaturing gels revealed that a small proportion of U2 small nuclear ribonucleoprotein (snRNP) displays a low mobility, in confirmation of previous reports. This low mobility form of U2 snRNP (termed LMC, for low mobility complex) also formed in vitro when U2 snRNP present in HeLa cytoplasmic S100 was added to a micrococcal nuclease-treated nuclear extract. Of greater experimental value, we found that the LMC also formed when a T7 U2 RNA transcript was assembled into U2 snRNP in a HeLa cytoplasmic S100 system, followed by its incubation in micrococcal nuclease-treated nuclear extract. LMC formation was ATP-dependent and was specific for U2 snRNP since it was not observed with S100-assembled U1 or U4 snRNPs. RNase H cleavage of U2 snRNP in the nuclear extract with an oligonucleotide complementary to nucleotides 28-42 of U2 RNA, as opposed to micrococcal nuclease treatment, rendered the extract competent to form the LMC, indicating that the nuclear factors responsible for LMC formation reside on endogenous U2 snRNP. LMC formation was not competed by excess U2 RNA but was competed by partially purified native U2 snRNP, providing further evidence that the LMC represents an interaction of nuclear factors with already assembled U2 snRNP. LMC formation did not take place on a mutant U2 snRNP lacking the binding site for the two U2-specific proteins, A' and B", nor on mutant U2 snRNPs lacking nucleotides 34-37 or nucleotides 46-49. Further results revealed that nucleotides 35 and 36 of U2 RNA, but not 34 and 37, are required for LMC formation. These experiments demonstrate a nucleotide sequence-specific interaction of U2 snRNP with nuclear factors in the absence of pre-mRNA. Among the U2 RNA nucleotides involved in the formation of this complex are ones previously implicated in base pairing between U2 RNA and the pre-mRNA lariat branch site. These findings are discussed in the context of the possibility that the LMC is on the spliceosome assembly pathway.