PB1-F2 is a pro-apoptotic polypeptide of many influenza A virus (FLUAV) isolates encoded by an alternative ORF of segment 2. A comprehensive GenBank search was conducted to analyse its prevalence. This search yielded 2226 entries of 80 FLUAV subtypes. Of these sequences, 87 % encode a PB1-F2 polypeptide greater than 78 aa. However, classic swine influenza viruses and human H1N1 isolates collected since 1950 harbour a truncated PB1-F2 sequence. While PB1-F2 of human H1N1 viruses terminates after 57 aa, classic swine H1N1 sequences have in-frame stop codons after 11, 25 and 34 codons. Of the avian sequences, 96 % encode a full-length PB1-F2. One genetic lineage of segment 2 sequences which is avian-like and different from the classic swine FLUAV comprises PB1-F2 sequences of porcine FLUAVs isolated in Europe (H1N1, H1N2, H3N2). Of these PB1-F2 sequences, 42 % also exhibit stop codons after 11, 25 and 34 codons. These amino acid positions are highly conserved among all FLUAV isolates irrespective of their origin. Molecular genetic analyses reveal that PB1-F2 is under constraint of the PB1 gene. The PB1-F2 polypeptide of FLUAVs isolated from European pigs is expressed in host cells as demonstrated by immunohistochemistry. Using different PB1-F2 versions fused to an enhanced GFP, mitochondrial localization is demonstrated for those PB1-F2 polypeptides which are greater than 78 aa while a truncated version (57 aa) shows a diffuse cytoplasmic distribution. This indicates similar properties and function of porcine and human FLUAV PB1-F2.