After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.