The hnRNP C1 and C2 proteins are among the most abundant proteins in the nucleus, and as ubiquitous components of RNP complexes, they have been implicated in many aspects of mRNA biogenesis. In this report, we have characterized a null mutation induced in embryonic stem cells by insertion of the U3His gene trap retrovirus into the first intron of the hnRNP C1/C2 gene. cDNAs encoding murine hnRNP C1 and C2 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. A human consensus sequence, generated from over 400 expressed sequence tags, suggests two revisions to the previously published human sequence. In addition, alternatively spliced transcripts, expressed only by the murine gene, encode four novel proteins: variants of C1 and C2 with either seven additional amino acids or one fewer amino acid in a region between the oligomerization and C-terminal acidic domains. The disrupted gene was transmitted into the germ line and is tightly linked to a recessive, embryonic lethal phenotype. Homozygous mutant embryos fail to develop beyond the egg cylinder stage and are resorbed by 10.5 days of gestation, a phenotype consistent with a fundamental role in cellular metabolism. However, hnRNP C1 and C2 are not required for cell viability. Embryonic stem cell lines established from homozygous mutant blastocysts did not express detectable levels of either protein yet were able to grow and differentiate in vitro, albeit more slowly than wild-type cells. These results indicate that the C1 and C2 hnRNPs are not required for any essential step in mRNA biogenesis; however, the proteins may influence the rate and/or fidelity of one or more steps.