The 25-kDa mRNA cap-binding protein (CBP) involved in translation was purified by affinity chromatography from human erythrocytes and rabbit reticulocytes. The sequences of eight human and seven rabbit tryptic and V8 proteolytic peptides were determined. Based on the peptide sequence data, oligodeoxynucleotide probes were synthesized and used to screen human fibroblast and lymphocyte lambda cDNA libraries. The DNA sequence obtained from recombinant lambda phage inserts was found to code for all but one peptide. A 23-base oligonucleotide was synthesized based on the DNA sequence and used to prime synthesis of cDNA from human placental mRNA to construct a third library in lambda gt10. Screening with a 22-base oligonucleotide, whose sequence was upstream from the 23-base primer, yielded numerous recombinant phages with approximately equal to 250-base inserts. The 1900-base-pair cDNA sequence compiled from all phage inserts appeared to represent the entire primary sequence of CBP (Mr 25,117). Blot analysis of human placental and HeLa mRNA revealed multiple CBP mRNA species ranging from 1925 to 2250 bases. The amino acid sequence of CBP showed homology to the cap-binding PB2 protein of influenza virus.