Recruitment of the U2 snRNP to the pre-mRNA is an essential step in spliceosome assembly. Although the protein components of the U2 snRNP have been identified, their individual contributions to function are poorly defined. In vitro studies with the Drosophila and human proteins suggest that two of the U2 snRNP-specific proteins, U2A' and U2B", function exclusively as a dimer. In Drosophila the presence of the U2B" counterpart, Sans-Fille (SNF), in the U2 snRNP is dispensable for viability, suggesting that SNF is not necessary for U2 snRNP function in vivo. With the identification of a single U2A'-like protein in the Drosophila genome, we can now investigate the relationship between SNF and its putative binding partner in vivo. Here we show that Drosophila U2A' protein interacts with SNF in vivo and, like its human counterpart, is U2 snRNP specific. Unexpectedly, however, we find that loss of function causes lethality, suggesting that U2A', but not SNF, is critical for U2 snRNP function. Moreover, although we demonstrate that several domains in the SNF protein are important for the interaction with the Drosophila U2A' protein, including a redundant domain at the normally dispensable C-terminus, we find that U2A' does not require heterodimer formation for either its vital function or U2 snRNP assembly. Thus together these data demonstrate that in Drosophila U2A' has an essential function that is unrelated to its role as the partner protein of SNF/U2B".