The fiber gene of adenovirus type 41 was sequenced and compared to the fiber gene sequence of adenovirus type 40 (A. H. Kidd and M. J. Erasmus, 1989, Virology 172, 134-144), the other known member of subgroup F. The open reading frame, from map units 87 through 92 with transcription from the r-strand, comprised 1686 bases and was 45 bases longer than its counterpart on the Ad40 genome. The 45-base difference appears to have resulted from a block deletion on the Ad40 sequence. Apart from this one region, the Ad40 and Ad41 fiber genes showed remarkably high homology (95.6%), indicating a relatively recent evolutionary divergence. The deduced amino acid sequence of the Ad41 fiber polypeptide was analyzed according to the model of N. M. Green et al. (1983, EMBO J. 2, 1357-1365) for the structure of the adenovirus fiber. Ad41 had one more 15-residue repeat in the shaft region than Ad40, there being 22 repeat motifs. A detailed study of various Ad40 and Ad41 strains with proven genome differences indicated that the 15-amino acid difference in polypeptide length at the 14th repeat motif is a type-specific difference among the subgroup F adenoviruses. However, two uncommon Ad41 strains belonging to 2 of the 16 Ad41 genome types tested had a 15-amino acid block deletion which was different to that of the Ad40 polypeptide. The implication from this work is that the Ad40 fiber gene probably arose from its Ad41 counterpart, but the fiber gene sequences of both types of subgroup F adenovirus are so similar that genetic recombination between strains could occur with some frequency.