A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.